And (-)-Cannabidiol

SUPPLEMENTAL MATERIAL

1. Materials and methods

Materials

AM251, AM630, anandamide (AEA), and (-)-cannabidiol (CBD) were purchased from Cayman Chemical Company; AMG 9810, γ-irradiated lipopolysaccharides from Escherichia coli 026:B6 (LPS), lipoteichoic acid from Staphylococcus aureus (LTA), arachidonic acid (AA), capsazepine (CPZ), cyclosporine A (CSA), GF109203X (GF), GSK1016790A (GSK), H89, linoleic acid (LA), ruthenium red (RR) and testosterone (T) were obtained from Sigma-Aldrich. HC067047 (HC) was purchased from Maybridge Ltd., ZM241385 (ZM) from Tocris Bioscience, and Gö6976 (Gö) and wortmannin (WM) from Calbiochem. AA, AEA, AM251, CBD and LA were dissolved in absolute ethanol (Sigma-Aldrich), while the solvent for AM630, AMG 9810, CBD (only in hSOC experiments), CPZ, CSA, GF, Gö, GSK, HC, RR, T, WM and ZM was DMSO (Sigma-Aldrich). LPS, LTA and H89 were dissolved in filtered distilled water.

Cell culturing

Human immortalized SZ95 sebocytes, originated from human facial sebaceous glands (19), were cultured in Sebomed® Basal Medium (Biochrom) supplemented with 10% fetal bovine serum (LifeTechnologies), 1 mM CaCl2, 5 ng/ml human epidermal growth factor (Sigma-Aldrich), 50 IU/ml penicillin and 50 μg/ml streptomycin (both from Teva). The final calcium concentration of the medium was approximately 1.25 mM (“high-Ca2+ medium”). The “low-Ca2+ Sebomed medium” was prepared to set the Ca2+ concentration to 0.25 mM. The medium was changed every other day, and cells were sub-cultured at 60-70% confluence.

Determination of intracellular lipids

For semi-quantitative detection of sebaceous lipids, cells were cultured on glass coverslips, and treated with various compounds for 24 hrs. Cells were then washed in phosphate-buffered saline (PBS; 115 mM NaCl, 20 mM Na2HPO4, pH 7.4; all from Sigma-Aldrich), fixed in 4% paraformaldehyde/PBS (Sigma-Aldrich), washed again twice in PBS and once in 60% isopropanol, and stained in freshly prepared Oil Red O solution (in 60% isopropanol) (Sigma-Aldrich) for 20 minutes at 37°C. Nuclei were counterstained with Mayer’s hematoxylin (Sigma-Aldrich) for 20 s and coverslips were finally mounted in mounting medium (DAKO).

For quantitative measurement of lipid content, cells (densities: 2,000 cells/well [6-day treatments] or 20,000 cells/well [24-48-hr treatments]) were cultured in 96-well black-

1 well/clear-bottom plates (Greiner Bio-One) in quadruplicates, and were treated with compounds as indicated. Subsequently, supernatants were discarded, cells were washed twice with PBS, and 100 μl of a 1 μg/ml Nile Red (Sigma-Aldrich) solution in PBS was added to each well. The plates were then incubated at 37°C for 20 min, and fluorescence was measured on a Molecular Devices FlexStation384 II or FlexStation 3 FLuorescence Image MicroPlate Reader (FLIPR; Molecular Devices). Results are expressed as percentages of the relative fluorescence units (RF) in comparison with the controls using 485 nm excitation and 565 nm emission wavelengths for neutral lipids, and 540 nm excitation and 620 nm emission wavelengths for polar lipids.

Investigation of the lipidome

Chemicals Eluents and extraction solvents were of HPLC/MS grade and were purchased from Merck. Ammonium formate (HCOONH4) was of HPLC/MS grade and was purchased in granular form from Fluka. The authentic triglycerides (TG) 1-palmitoyl-2-oleoyl-3- lineoleoyl-rac-glycerol and 1,3-dipalmitoyl-2-oleoylglycerol were purchased from Cayman Chemical Company and Sigma-Aldrich, respectively. The authentic diacylglyceride (DG) 1,2-dioleoyl-sn-glycerol was purchased from Cayman. The authentic free fatty acids (FFA) linoleic, palmitoleic, palmitic, stearic, oleic, linolenic, and arachidonic acids, as well as the standard wax ester (WE) lauryl palmitoleate, and the standard squalene (SQ), free cholesterol (CH) and cholesteryl esters (CE) oleate, linoleate, and arachidonate were purchased from Sigma-Aldrich. Dodecanoyl sphingomyelin (12:0 SM) was purchased from Avanti Polar Lipids, and used as the internal standard.

Sample extraction SZ95 sebocytes were harvested by trypsinization and counted. Before extraction, cell pellets were suspended in 500 µl of 5 mM ammonium formate. Extraction of neutral lipids was performed as previously described with slight modifications (78). 500 µl of abs. EtOH and 500 pM of the internal standard 12:0 SM were added to the cell suspension and mixed thoroughly. Liquid-liquid extraction was performed twice with 3 ml EtOAc. The unified layers of EtOAc were evaporated under a gentle stream of nitrogen. The total lipid extract was dissolved in Ac2O/MeOH/iPrOH 40/40/20 before injection.

LC-MS analysis of lipid extracts The lipid extracts were analyzed as previously reported (78). Shortly, the rapid resolution reversed phase HPLC (RR-RP-HPLC) separation was performed with Zorbax SB-C8 stationary phase. Lipids were eluted with a binary gradient of (A) 5 mM ammonium formate in MilliQ water (18.2 ) and (B) MeOH/iPrOH 95/5. The eluent outlet was connected to a G6220A series time of flight mass spectrometer (ToF-MS, Agilent Technologies) by means of electrospray ionization (ESI) interface

2 operating in the positive and negative ion modes. Neutral lipids and free fatty acids were detected in the positive and the negative ion mode, respectively. Scan mode ToF mass spectra were acquired in the positive and negative ion mode by using the ToF at 10,000 mass resolving power for scans over the range from m/z 100 to m/z 1000. MS scans were processed using the Mass Hunter software (B.01.03 version). The resulting data were converted to mass centroid from which the accurate m/z value was measured. Accurate mass measurements and isotopic patterns were the basis to retrieve the elemental composition and unsaturation degree of detected compounds. Identity of lipid species was established with the previously reported methodology (78).

Data extraction and statistical analysis Following the RR-RP-HPLC separation, retention times of neutral and acidic lipids that were expected to be present in the SZ95 sebocyte lipid extracts were consistent with those of authentic standards and with our previous findings. Peak areas of the individual lipids detected in the positive and negative ion modes were obtained from extracted ion chromatograms (EIC) derived by extraction of the m/z values of their pseudomolecular ion (78). To determine the relative abundance of neutral lipids the peak area of individual lipid species detected in the positive ion mode was normalized by the cell number and the peak area of the internal standard 12:0 SM in the EIC obtained by extraction of the m/z 647.5128 corresponding to the [M+H]+ ion C35H72N2O6P. To determine the relative abundance of FFA, which were detected in the negative ion mode, their peak area was normalized by the cell number and the peak area of the internal standard 12:0 SM in the EIC obtained by extraction of the m/z 691.5032 corresponding to the [M+HCOO]- ion C36H72N2O8P of the internal standard. Total relative abundance of lipid groups, i.e. TG, DG, WE, CE, and FFA was obtained by summing the relative abundance of individual lipids belonging to the same lipid class.

Determination of cellular viability

The viability of the cells was determined by measuring the conversion of the tetrazolium salt MTT (Sigma-Aldrich) to formazan by mitochondrial dehydrogenases. Cells were plated in 96-well plates (densities: 2,000 cells/well [6-day treatments] or 20,000 cells/well [2-day treatments]) in quadruplicates, and were cultured for 2 or 6 days. Cells were then incubated with 0.5 mg/ml MTT for 2 hrs, and concentration of formazan crystals (as an indicator of number of viable cells) was determined colorimetrically as described previously (10, 12). Results were expressed as percentage of vehicle controls regarded as 100%.

Determination of apoptosis

A decrease in the mitochondrial membrane potential is one of the earliest markers of apoptosis. Therefore, to assess the process, mitochondrial membrane potential of

SZ95 sebocytes was determined using a MitoProbe™ DiIC1(5) Assay Kit (Life Technologies). Cells (20,000 cells/well) were cultured in 96-well black-well/clear- bottom plates (Greiner Bio One) in quadruplicates and were treated with various compounds for 24 hrs. After removal of supernatants, cells were incubated for 30 minutes with DilC1(5) working solution (50 μl/well), then washed with PBS, and the fluorescence of DilC1(5) was measured at 630 nm excitation and 670 nm emission wavelengths using the above FLIPRs (Molecular Devices). RF values were expressed as percentage of vehicle controls regarded as 100%.

Determination of necrosis

Necrotic processes were determined by SYTOX Green staining (Life Technologies). The dye is able to penetrate (and then bind to the nucleic acids) only to necrotic cells with ruptured plasma membranes, whereas healthy cells with intact surface membranes show negligible SYTOX Green staining. Cells were cultured in 96-well black-well/clear-bottom plates (Greiner Bio One), and treated with CBD for up to 24 hrs. Supernatants were then discarded, and the cells were incubated for 30 minutes with 1 μM SYTOX Green dye. Following incubation, cells were washed with PBS, the culture medium was replaced, and fluorescence of SYTOX Green was measured at 490 nm excitation and 520 nm emission wavelengths using FLIPR (Molecular Devices).

Determination of cellular proliferation

The degree of cellular growth (reflecting proliferation) was determined by measuring the DNA content of cells using CyQUANT Cell Proliferation Assay Kit (Life Technologies). SZ95 sebocytes (5,000 cells per well) were cultured in 96-well black- well/clear-bottom plates (Greiner Bio-One) and were treated as indicated for 24, 48 and 72 hrs. Supernatants were then removed by blotting on paper towels, and the plates were subsequently frozen at -80°C. The plates were then thawed at room temperature, and 200 μl of CyQUANT dye/cell lysis buffer mixture was added to each well. After 5 minutes of incubation, fluorescence was measured at 490 nm excitation and 520 nm emission wavelengths using FLIPR (Molecular Devices).

RNA isolation, reverse transcription, quantitative “real-time” PCR (Q-PCR)

Q-PCR was performed on an ABI Prism 7000 sequence detection system (Applied Biosystems) or Stratagene Mx3005P QPCR System (Agilent Technologies) using the 5’ nuclease assay. Total RNA was isolated using TRIzol (LifeTechnologies), DNase

4 treatment was performed according to the manufacturer’s protocol, and then 1 μg of total RNA were reverse-transcribed into cDNA by using 15 IU of AMV reverse transcriptase (Promega) and 0.025 μg/μl random primers (Promega). PCR amplification was performed by using the TaqMan primers and probes (assay ID-s: Hs00189038_m1 for cathelicidin, Hs00174097_m1 for IL1B, Hs00985639_m1 for IL6, Hs01032443_m1 for Ki67 (MKI67), Hs00942766_s1 for NRIP1, Hs01082394_m1 for TRIB3, Hs00261256_m1 for ARHGAP9, Hs00169123_m1 for A2a receptor (ADORA2A), Hs00174128_m1 for TNFA, Hs00218912_m1 for TRPV1, Hs00275032_m1 for TRPV2, Hs00222101_m1 for TRPV4, Hs00175798_m1 for TRPA1 and Hs00375481_m1 for TRPM8) and the TaqMan universal PCR master mix protocol (Applied Biosystems). As internal controls, transcripts of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), peptidyl-prolyl isomerase A (cyclophilin A; PPIA), and 18S ribosomal RNA (18S) were determined (assay ID-s: Hs99999905_m1, Hs 99999904_m1, and Hs99999901_s1, respectively). The amount of the transcripts was normalized to those of the relevant housekeeping gene using the ΔCT method. When indicated, the results were then normalized to the expression of the vehicle control (ΔΔCT method).

Immunocytochemistry

SZ95 sebocytes, seeded and cultured on sterile coverslips in 24-well plates, were fixed in ice-cold acetone for 5 min and then permeabilized by 0.1% Triton-X-100 (Sigma-Aldrich) in PBS for 10 min. After washing in PBS and blocking in 1% BSA (Sigma-Aldrich) in PBS for 30 min, cells were incubated with the TRPV1 (Sigma- Aldrich), TRPV2, TRPA1, TRPM8, A2a (AbCam) and TRPV4 (Alomone Labs) specific primary antibodies (all produced in rabbit; dilution 1:500 in blocking solution; overnight incubation at 4°C). For fluorescence staining, slides were then incubated with Alexa-Fluor®-488-conjugated rabbit IgG Fc segment-specific secondary antibodies (developed in goat; Life Technologies) for 60 min (dilution 1:200), and the nuclei of cells were visualized using DAPI (Vector Laboratories). Cells were examined on a Nikon Eclipse E600 fluorescent microscope (Nikon). As negative controls, the appropriate primary antibodies were omitted from the procedure.

Western blotting

Cells were harvested in lysis buffer (20 mM Tris-HCl, pH 7.4, 5 mM EGTA, 1 mM 4- (2-aminoethyl)benzensulfonyl fluoride, protease inhibitor cocktail diluted 1:100, all from Sigma-Aldrich) and the protein content was measured by a modified BCA protein assay (Pierce). The samples were then subjected to sodium dodecyl sulfate- polyacrylamide gel electrophoresis (7.5% gels were loaded with equal [20-60 μg] amount of protein per lane), transferred to BioBond nitrocellulose membranes (Whatman), and then probed with rabbit-anti-human NRIP1 and TRIB3 (both from

Novus Biologicals; 1:200 dilution in 5% milk containing PBS), rabbit-anti-human MAPK ERK1/2, mouse-anti-human P-ERK1/2 (both from Santa Cruz; 1:1500 dilution in both cases in 5% milk containing PBS), mouse-anti-human P-IκBα (Cell Signaling; 1:1000 dilution in 5% milk containing PBS), rabbit-anti-P-P65 (Novus Biologicals; 1:1000 dilution in 5% milk containing PBS) or the above primary anti-TRP channel and anti-A2a receptor specific antibodies (all of them were applied in 1:200 in 5% milk containing PBS). As secondary antibodies, horseradish peroxidase-conjugated rabbit or mouse IgG Fc segment-specific antibodies (developed in goat and sheep, respectively, 1:1000, Bio-Rad) were used, and the immunoreactive bands were visualized by a SuperSignal® West Pico Chemiluminescent Substrate enhanced chemiluminescence kit (Pierce) using a KODAK Gel Logic 1500 Imaging System (Eastman Kodak Company). To assess equal loading, when indicated, membranes were re-probed with anti-β-actin antibodies and visualized as described above.

Semiquantitative densitometric analysis of the signals was performed by using Image J software (NIH).

Full-thickness human skin organ culture and sample preparations

Sample preparation and organ culture

4 mm biopsies of intact human scalp and arm skin samples obtained from 4 female individuals (average age: 56 years old) were punched out using a biopsy punch (pfm medical). These biopsies were maintained in serum-free William’s E medium (Biochrom) supplemented with 2 mM L-glutamine (Life Technologies), 10 ng/ml hydrocortisone (Sigma-Aldrich), 10 μg/ml insulin (Sigma-Aldrich), and 1% antibiotic/antimycotic mixture (PAA Laboratories GmbH). The control group received vehicle alone. The skin biopsies were left to float in the medium, with the epidermis up at the air/liquid interface and the dermis/subcutis down (20). The cultures were incubated at 37°C in a gassed incubator with 95% air and 5% CO2 at 37°C.

Oil Red O staining

After the organ culture, skin samples were embedded in Cryomatrix (Thermo Shandon Limited) and frozen in liquid nitrogen. Cryo-sections of 6 μm thickness were processed for Oil red O histochemistry. After washing in distilled water, cryo-sections were incubated in 60% isopropanol (Sigma-Aldrich), and stained in freshly prepared Oil Red O solution (0.3% in isopropanol; Sigma-Aldrich). Nuclei were counterstained with hematoxylin (Carl Roth GmbH) and the sections were mounted in aqueous mounting medium (DAKO). Images were analyzed by Image J image analysis software (NIH).

MKI67 staining

To assess proliferation in human skin organ cultures, a MKI67 staining method was employed. Briefly, after CBD treatment, cryostat sections were labeled with a mouse anti-MKI67 antiserum (1:20, DAKO). MKI67-positive cells were visualized by using a rhodamine-conjugated goat anti-mouse secondary antibody (1:200, Jackson ImmunoResearch). Sections were then counterstained with DAPI (4’,6-diamidino-2- phenylindole; Vector Laboratories). The analysis for cell counting on 2 sections per group was performed using a fluorescence microscope BZ-8100 (Biozero, Keyence). The distance between two analyzed sections was more than 50 μm.

RNA interference (RNAi)

RNAi was performed according to our optimized protocols (10, 38). In brief, SZ95 sebocytes at 50-70% confluence were transfected with specific Stealth RNAi oligonucleotides (40 nM) against NRIP1 (ID-s: HSS112045 (“siNRIP1a”) and HSS112046 (“siNRIP1b”)), TRIB3 (ID-s: HSS184051 (“siTRIB3a”) and HSS184052 (“siTRIB3b”), TRPV1 (ID-s: HSS111304 (“sV1a”) and HSS111306 (“siV1b”)), TRPV2 (ID-s: HSS122144 (“siV2a”) and HSS122145 (“siV2b”)) and TRPV4 (ID-s: HSS126973 (“siV4a”) and HSS126974 (“siV4b”)) using Lipofectamine 2000 (all from Life Technologies). For controls, RNAi Negative Control Duplexes (Scrambled RNAi “medium”) were applied. The efficacy of RNAi-driven “knock-down” was evaluated daily by Western blotting and by Q-PCR.

Microarray analysis

Gene expression analysis of three independent sets of control and CBD-treated SZ95 sebocytes (10 μM CBD for 24 hrs) was performed by using Human Whole Genome Oligo Microarray® (44K) (Agilent Technologies). Total RNA was isolated using TRIzol (LifeTechnologies) according to the manufacturer’s protocol, and the isolated RNA was quality-checked by Agilent 2100 Bioanalyzer platform (Agilent Technologies) and Nanodrop-1000 Spectrophotometer (NanoDrop/Thermo Scientific). Alterations in the gene expression were regarded as significant if (i) there were at least two-fold changes in the corresponding levels; (ii) the changes were equi-directional in all cases; and (iii) global, corrected P value was less than 0.05. Evaluation, Gene Set Enrichment Analysis (GSEA) and Biological Networks Gene Ontology analysis (BiNGO) of the results was performed by Abiomics Ltd (http://www.abiomics.eu).

Data have been deposited in NCBI's Gene Expression Omnibus (79) and are accessible through GEO Series accession number GSE57571 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE57571).

Determination of the intracellular 3’-5’-cyclic adenosine monophosphate concentration (cAMP ELISA)

SZ95 sebocytes were treated for 1 hour with vehicle or CBD (10 μM). Cells were then lysed (cell density: 107 cells/ml), and cell lysates were assayed immediately according to the manufacturer’s protocol, using Parameter Cyclic AMP Assay (R&D Systems). Evaluation of the data was performed by using “Four Parameter Logistic Curve” online data analysis tool of MyAssays Ltd. (http://www.myassays.com/four- parameter-logistic-curve.assay).

Patch-clamp recording

Whole cell patch clamp measurements were made by using an Axopatch 200A amplifier and Clampex 10.0 software (Molecular Devices) or an EPC-10 amplifier and Patchmaster software (HEKA Elektronik). To record CBD evoked transmembrane currents, experiments were performed in normal Ringer solution (in mM: NaCl, 140;

KCl, 5; glucose, 10; HEPES, 10; CaCl2, 2; MgCl2, 1; and sodium-pyruvate, 1; pH 7.2) and patch pipettes were filled with a solution containing (in mM): K-gluconate, 120; NaCl, 5; 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid (HEPES), 10; EGTA, 2;

CaCl2, 0.1; Mg-ATP, 5; Na3-GTP, 0.3; Na2-phosphocreatinine, 10; pH 7.3. To record TRPV4 current, the bath solution consisted of 150 mM NaCl, 6 mM CsCl, 5 mM

CaCl2, 1 mM MgCl2, 10 mM HEPES and 10 mM glucose buffered to pH 7.4 (NaOH), whereas the pipette solution consisted of 100 mM aspartic acid, 20 mM CsCl, 1 mM

MgCl2, 0.08 mM CaCl2, 4 mM Na2ATP, 10 mM BAPTA, 10 mM HEPES and pH was set to 7.2 using CsOH resulted in ca. 100 mM CsAsp in the final pipette solution. The holding potential was 0 mV and cells were ramped every 2 s from −120 to +100 mV over the course of 400 ms.

Fluorescent Ca2+-imaging

Fluorescent Ca2+-imaging was performed according to our previously optimized protocol (80). In brief, SZ95 sebocytes were seeded in 96-well black-well/clear- bottom plates (Greiner Bio-One) at a density of 20,000 cells per well. Cells were washed two times with 1% bovine serum albumin (Sigma-Aldrich) and 2.5 mM Probenecid (Sigma-Aldrich) containing Hank’s solution (136.8 mM NaCl, 5.4 mM KCl,

0.34 mM Na2HPO4, 0.44 mM KH2PO4, 0.81 mM MgSO4, 1.26 mM CaCl2, 5.56 mM glucose, 4.17 mM NaHCO3, pH 7.2, all from Sigma-Aldrich). The cells were then incubated with 1 μM Fluo-4 AM (Life Technologies) containing Hank’s solution (100 μl/well) at 37°C for 30 min, and were then washed three times with Hank’s solution 2+ (100 μl/well; in the case of the “low Ca Hank” the CaCl2 content was supplemented by equimolar glucose). The plates were then placed in a FLIPR (Molecular Devices), 2+ and changes in [Ca ]IC (reflected by changes fluorescence; excitation: 490 nm;

8 emission: 520 nm) induced by various concentrations of the compounds were recorded in each well. Experiments were performed in triplicates and the averaged data (as well as SEM) were used in the calculations.

Statistical analysis

Data were analyzed by IBM SPSS Statistics 19 (SPSS Inc.) software, using Student’s two tailed two samples t-test (paired comparisons) or one-way ANOVA with Bonferroni’s and Dunnett’s post hoc probes (multiple comparisons) and P<0.05 values were regarded as significant differences. Homogeneity of variances were analyzed by Levene’s test. If Levene’s test indicated inhomogeneity of variances, Games-Howel test was used instead of Bonferroni. Graphs were plotted by using Origin Pro Plus 6.0 software (Microcal).

Study approval

This study was approved by the Institutional Research Ethics Committee (University of Lübeck) and adhered to the Declaration of Helsinki Principle guidelines. Study subjects provided informed consent prior to their participation.

2. Supplemental Tables

Supplemental Table 1. List of genes that were significantly down-regulated (3 independent treatments, at least 2-fold equi-directional changes in all cases, P<0.05) by CBD treatment of human SZ95 sebocytes

Average Gene Name Description Note Fold Change Homo sapiens midline 1 (Opitz/BBB MID1 syndrome) (MID1), transcript variant 3, 0.144583754 mRNA [NM_033290] Same effect was Homo sapiens solute carrier family 39 shown in BV-2 SLC39A10 (zinc transporter), member 10 0.179378308 microglia cells; (SLC39A10), mRNA [NM_020342] SLC39A10 is involved in Zn2+-efflux (81). ENST00000380787 Unknown 0.218302567 Homo sapiens N-acetyltransferase 1 NAT1 (arylamine N-acetyltransferase) 0.220518435 (NAT1), mRNA [NM_000662] Homo sapiens CD24 signal transducer CD24 mRNA, complete cds and 3' region. 0.244703822 [L33930] A_32_P82293 Unknown 0.276372603 Homo sapiens cDNA FLJ27066 fis, ARHGAP24 0.286928365 clone SPL01327. [AK130576] It is thought to function as a Homo sapiens disheveled associated scaffolding protein for DAAM1 activator of morphogenesis 1 0.288870651 the Wnt-induced (DAAM1), mRNA [NM_014992] assembly of a disheveled (Dvl)-Rho complex (82). Homo sapiens opsin 3 (encephalopsin, OPN3 panopsin) (OPN3), transcript variant 1, 0.294213681 mRNA [NM_014322] Homo sapiens laminin, beta 1 LAMB1 0.295427431 (LAMB1), mRNA [NM_002291] Homo sapiens netrin 4 (NTN4), mRNA It promotes cellular NTN4 0.297136326 [NM_021229] proliferation (83). UI-H-BI4-apz-e-08-0-UI.s1 NCI_CGAP_Sub8 Homo sapiens BF508144 0.298223395 cDNA clone IMAGE:3089007 3', mRNA sequence [BF508144] Homo sapiens protease, serine, 23 PRSS23 0.301106646 (PRSS23), mRNA [NM_007173] Homo sapiens STEAP family member It is thought to be STEAP4 0.303239595 involved in adipocyte 4 (STEAP4), mRNA [NM_024636] differentiation (84). Homo sapiens cDNA FLJ90790 fis, clone THYRO1001529, moderately SPTLC2L 0.304280871 similar to Serine palmitoyltransferase 2 (EC 2.3.1.50). [AK075271] Homo sapiens forkhead box A1 It is a mesenchymal FOXA1 0.304794447 differentiation marker (FOXA1), mRNA [NM_004496] (85).

Homo sapiens latexin (LXN), mRNA LXN 0.309134992 [NM_020169] Homo sapiens nuclear receptor Essential for NRIP1 interacting protein 1 (NRIP1), mRNA 0.309212846 triglyceride storage in [NM_003489] adipose tissue (46). Homo sapiens toll-like receptor 1 Role in innate TLR1 0.312672783 (TLR1), mRNA [NM_003263] immunity (86). ENST00000353442 Unknown 0.314827882 It plays a role in the Homo sapiens mitogen-activated activation of mTORC1 MAP4K3 protein kinase kinase kinase kinase 3 0.325163457 (87), which is thought (MAP4K3), mRNA [NM_003618] to have “pro-acne” effects (43). Homo sapiens translocation TRAM1 associated membrane protein 1 0.327653171 (TRAM1), mRNA [NM_014294] THC2386010 Unknown 0.331284758 Homo sapiens thiopurine S- TPMT methyltransferase (TPMT), mRNA 0.332831935 [NM_000367] Homo sapiens LIM domain 7 (LMO7), LMO7 0.334474202 mRNA [NM_005358] Q9N3X9 (Q9N3X9) Collagen protein THC2367825 0.335682775 115, partial (5%) [THC2367825] Homo sapiens mRNA; cDNA ENST00000367385 DKFZp586G2222 (from clone 0.336935039 DKFZp586G2222). [AL080111] ALU8_HUMAN (P39195) Alu subfamily SX sequence contamination THC2279364 0.337174074 warning entry, partial (4%) [THC2279364] AV698092 GKC Homo sapiens cDNA AV698092 clone GKCGUA09 5', mRNA sequence 0.344919878 [AV698092] Homo sapiens full length insert cDNA AF086216 0.344946688 clone ZC65B11. [AF086216] Homo sapiens cDNA FLJ12985 fis, clone NT2RP3000050, moderately ZNF702 0.349667263 similar to ZINC FINGER PROTEIN 91. [AK023047] Homo sapiens EGF-containing fibulin- like extracellular matrix protein 1 EFEMP1 0.350370379 (EFEMP1), transcript variant 1, mRNA [NM_004105] PREDICTED: Homo sapiens similar to general transcription factor II, i isoform LOC649791 0.351190156 1, transcript variant 4 (LOC649791), mRNA [XM_943120] Homo sapiens KIAA0101 (KIAA0101), KIAA0101 transcript variant 1, mRNA 0.353153053 [NM_014736] Homo sapiens POT1 protection of POT1 telomeres 1 homolog (S. pombe) 0.35484116 (POT1), mRNA [NM_015450] Homo sapiens clone FLB1727 AF113674 PRO0398 mRNA, complete cds. 0.359119921 [AF113674]

Homo sapiens hypothetical protein MGC12538, mRNA (cDNA clone RP4-747L4.3 0.359262384 MGC:12538 IMAGE:3839075), complete cds. [BC007072] Homo sapiens Kruppel-like factor 5 It is pro-proliferative in KLF5 (intestinal) (KLF5), mRNA 0.360155512 epithelial cells (88). [NM_001730] Homo sapiens PNAS-12 mRNA, ENST00000297145 0.361030612 partial sequence. [AF274937] Homo sapiens chromosome 6 open C6orf211 reading frame 211 (C6orf211), mRNA 0.365046799 [NM_024573] Homo sapiens cDNA FLJ31683 fis, AK056245 0.366099279 clone NT2RI2005353. [AK056245] Homo sapiens phosphorylase kinase, PHKB beta (PHKB), transcript variant 1, 0.36678392 mRNA [NM_000293] UI-H-BI4-aoz-b-08-0-UI.s1 NCI_CGAP_Sub8 Homo sapiens BF509482 0.370735322 cDNA clone IMAGE:3086535 3', mRNA sequence [BF509482] Homo sapiens mRNA; cDNA AL832758 DKFZp686C0927 (from clone 0.371776581 DKFZp686C0927). [AL832758] Homo sapiens mRNA; cDNA ENST00000373218 DKFZp762K067 (from clone 0.372494994 DKFZp762K067). [CR627373] Homo sapiens phosphodiesterase 8A PDE8A (PDE8A), transcript variant 5, mRNA 0.377251072 [NM_173457] Homo sapiens family with sequence similarity 62 (C2 domain containing) FAM62B 0.378298525 member B (FAM62B), mRNA [NM_020728] Homo sapiens sciellin (SCEL), SCEL transcript variant 2, mRNA 0.382019937 [NM_144777] Homo sapiens nuclear transport factor NXT2 2-like export factor 2 (NXT2), mRNA 0.382153336 [NM_018698] Homo sapiens Rho guanine nucleotide ARHGEF3 exchange factor (GEF) 3 (ARHGEF3), 0.382355051 mRNA [NM_019555] BC004536 carnitine deficiency- associated gene expressed in ventricle THC2314901 0.382570549 1 {Homo sapiens;} , partial (7%) [THC2314901] Homo sapiens mRNA; cDNA AL133577 DKFZp434G0972 (from clone 0.383756041 DKFZp434G0972). [AL133577] Q62VJ0 (Q62VJ0) Small ENST00000367142 peptidoglycan-associated lipoprotein, 0.385407708 partial (13%) [THC2301029] Homo sapiens DEP domain containing DEPDC7 0.386647666 7 (DEPDC7), mRNA [NM_139160] Homo sapiens zinc finger protein 426 ZNF426 0.387439082 (ZNF426), mRNA [NM_024106]

Homo sapiens prenylcysteine oxidase PCYOX1 0.387560426 1 (PCYOX1), mRNA [NM_016297] It inhibits WNT- Homo sapiens secreted frizzled- signaling (89). WNT is SFRP1 related protein 1 (SFRP1), mRNA 0.392834472 an inhibitor of [NM_003012] sebaceous differentiation (90). Homo sapiens general transcription GTF2I factor II, i (GTF2I), transcript variant 1, 0.394530829 mRNA [NM_032999] Homo sapiens activity-dependent ADNP neuroprotector (ADNP), transcript 0.399327536 variant 1, mRNA [NM_015339] Homo sapiens supervillin (SVIL), SVIL transcript variant 2, mRNA 0.400968078 [NM_021738] 602338531F1 NIH_MGC_89 Homo BG163514 sapiens cDNA clone IMAGE:4446534 0.403181706 5', mRNA sequence [BG163514] Homo sapiens schlafen family member SLFN12 0.408594415 12 (SLFN12), mRNA [NM_018042] Homo sapiens ubiquitin-activating enzyme E1C (UBA3 homolog, yeast) UBE1C 0.409550306 (UBE1C), transcript variant 1, mRNA [NM_003968] Homo sapiens centromere protein I CENPI 0.416365112 (CENPI), mRNA [NM_006733] Homo sapiens FLJ22624 protein FLJ22624 0.41913823 (FLJ22624), mRNA [NM_024808] Homo sapiens high mobility group nucleosomal binding domain 3 HMGN3 0.424725991 (HMGN3), transcript variant 2, mRNA [NM_138730] Homo sapiens BTB (POZ) domain BTBD1 containing 1 (BTBD1), transcript 0.42599776 variant 1, mRNA [NM_025238] Homo sapiens cDNA FLJ11174 fis, ENST00000367612 0.432484207 clone PLACE1007367. [AK002036] Homo sapiens family with sequence similarity 96, member A (FAM96A), FAM96A 0.438190306 transcript variant 1, mRNA [NM_032231] Homo sapiens mRNA full length insert ENST00000366751 cDNA clone EUROIMAGE 2068962. 0.438266468 [AJ420589] Homo sapiens fyn-related kinase FRK 0.444706282 (FRK), mRNA [NM_002031] Homo sapiens nuclear receptor NCOA7 coactivator 7 (NCOA7), mRNA 0.445200315 [NM_181782] Homo sapiens transcription elongation factor A (SII)-like 1 (TCEAL1), TCEAL1 0.447935262 transcript variant 3, mRNA [NM_001006640] Homo sapiens serpin peptidase SERPINB9 inhibitor, clade B (ovalbumin), member 0.451637699 9 (SERPINB9), mRNA [NM_004155]

Homo sapiens collagen and calcium CCBE1 binding EGF domains 1 (CCBE1), 0.454814193 mRNA [NM_133459] Homo sapiens RNA binding motif RBM7 protein 7 (RBM7), mRNA 0.462986923 [NM_016090] Homo sapiens antigen identified by Proliferation marker MKI67 monoclonal antibody Ki-67 (MKI67), 0.474182345 (91). mRNA [NM_002417] ALU1_HUMAN (P39188) Alu subfamily J sequence contamination THC2342537 0.475837891 warning entry, partial (5%) [THC2342537] Homo sapiens GPI-anchored membrane protein 1 (GPIAP1), GPIAP1 0.490956422 transcript variant 2, mRNA [NM_203364] Homo sapiens RNA-binding protein FLJ20273 0.494577039 (FLJ20273), mRNA [NM_019027]

Supplemental Table 2. List of genes that were significantly (3 independent treatments, at least 2-fold equi-directional changes in all cases, P<0.05) up-regulated by CBD treatment of human SZ95 sebocytes

Average GeneName Description Note Fold Change Homo sapiens similar to RIKEN cDNA 2210021J22, transcript variant 1, mRNA LOC150383 (cDNA clone MGC:87534 2.023020498 IMAGE:30338205), complete cds. [BC067871] Homo sapiens cell death-inducing CICE CIDE-like effector pseudogene (CICE) 2.028682357 on chromosome 3 [NR_002786] PREDICTED: Homo sapiens LOC145853 hypothetical LOC145853 (LOC145853), 2.050746331 mRNA [XM_096885] THC2296299 Unknown 2.052834221 nj17d09.s1 NCI_CGAP_Pr22 Homo AA532655 sapiens cDNA clone IMAGE:986609 3', 2.100324768 mRNA sequence [AA532655] Homo sapiens cDNA FLJ42879 fis, SH2D5 2.10832994 clone BRHIP3001283. [AK124869] Homo sapiens cadherin 4, type 1, R- CDH4 cadherin (retinal) (CDH4), mRNA 2.109778874 [NM_001794] Homo sapiens HSPC254 mRNA, partial ENST00000269290 2.114503823 cds. [AF161372] Homo sapiens activating transcription factor 4 (tax-responsive enhancer It is a down-stream ATF4 2.138734073 molecule of TRIB3- element B67) (ATF4), transcript variant signaling (66). 1, mRNA [NM_001675] Homo sapiens glutamic-oxaloacetic transaminase 1, soluble (aspartate GOT1 2.1476287 aminotransferase 1) (GOT1), mRNA [NM_002079] Homo sapiens chromosome 6 open C6orf1 reading frame 1 (C6orf1), transcript 2.188775606 variant 1, mRNA [NM_178508] Homo sapiens solute carrier family 1 (glutamate/neutral amino acid SLC1A4 2.191168404 transporter), member 4 (SLC1A4), mRNA [NM_003038] Homo sapiens zinc finger protein 598 ZNF598 2.221106279 (ZNF598), mRNA [NM_178167] Homo sapiens ATP-binding cassette, sub-family B (MDR/TAP), member 6 ABCB6 (ABCB6), nuclear gene encoding 2.238983054 mitochondrial protein, mRNA [NM_005689] Homo sapiens fucosyltransferase 1 (galactoside 2-alpha-L- FUT1 2.24477072 fucosyltransferase, H blood group) (FUT1), mRNA [NM_000148]

Homo sapiens ST6 (alpha-N-acetylneuraminyl-2,3-beta-galactosyl-1,3)-N- acetylgalactosaminide alpha-2,6- ST6GALNAC4 2.249856903 sialyltransferase 4 (ST6GALNAC4), transcript variant 1, mRNA [NM_175039] Homo sapiens tryptophanyl-tRNA WARS synthetase (WARS), transcript variant 1, 2.262522437 mRNA [NM_004184] Homo sapiens MYB binding protein It is a repressor of MYBBP1A (P160) 1a (MYBBP1A), mRNA 2.285565522 NF-κB (92). [NM_014520] Homo sapiens cDNA FLJ41456 fis, AK123450 2.291105966 clone BRSTN2012320. [AK123450] Homo sapiens cDNA FLJ37088 fis, LOC645427 2.299131401 clone BRACE2017124. [AK094407] A_32_P146871 Unknown 2.325869291 Homo sapiens UbiA prenyltransferase UBIAD1 domain containing 1 (UBIAD1), mRNA 2.326335569 [NM_013319] Homo sapiens TIGA1 (TIGA1), mRNA It inhibits proliferation TIGA1 2.332263761 via maintaining G [NM_053000] 0 phase (93). Homo sapiens N-acetylglutamate NAGS 2.354736066 synthase (NAGS), mRNA [NM_153006] Homo sapiens solute carrier family 3 (activators of dibasic and neutral amino SLC3A2 acid transport), member 2 (SLC3A2), 2.372841331 transcript variant 3, mRNA [NM_002394] Homo sapiens seryl-tRNA synthetase SARS 2.3754084 (SARS), mRNA [NM_006513] Homo sapiens zinc finger, AN1-type ZFAND2A domain 2A (ZFAND2A), mRNA 2.395515359 [NM_182491] Homo sapiens cDNA FLJ26339 fis, AK129849 2.396346358 clone HRT02975. [AK129849] Homo sapiens phosphoglycerate PHGDH dehydrogenase (PHGDH), mRNA 2.401009252 [NM_006623] Homo sapiens ets variant gene 4 (E1A ETV4 enhancer binding protein, E1AF) 2.415031911 (ETV4), mRNA [NM_001986] xq40c08.x1 NCI_CGAP_Lu28 Homo sapiens cDNA clone IMAGE:2753102 3' AW275876 similar to gb:X57352 INTERFERON- 2.41597705 INDUCIBLE PROTEIN 1-8U (HUMAN);, mRNA sequence [AW275876] Homo sapiens glutamic pyruvate transaminase (alanine GPT2 2.418307046 aminotransferase) 2 (GPT2), mRNA [NM_133443] Homo sapiens La ribonucleoprotein domain family, member 6 (LARP6), LARP6 2.424631034 transcript variant 1, mRNA [NM_018357]

AL547890 AL547890 Homo sapiens PLACENTA COT 25-NORMALIZED THC2370432 Homo sapiens cDNA clone 2.436811494 CS0DI033YB09 5-PRIME, mRNA sequence [AL547890] A_24_P204414 Unknown 2.442940076 Homo sapiens ChaC, cation transport Down-stream of ATF4 CHAC1 regulator-like 1 (E. coli) (CHAC1), 2.449755082 (67). mRNA [NM_024111] Homo sapiens brain expressed X-linked BEX2 2.517811045 2 (BEX2), mRNA [NM_032621] Homo sapiens immunoglobulin IGSF4B superfamily, member 4B (IGSF4B), 2.535371171 mRNA [NM_021189] Homo sapiens serine dehydratase-like SDSL 2.538715595 (SDSL), mRNA [NM_138432] Homo sapiens cDNA: FLJ22719 fis, AK026372 2.555616463 clone HSI14307. [AK026372] Homo sapiens complement component C3AR1 3a receptor 1 (C3AR1), mRNA 2.570060685 [NM_004054] Homo sapiens homocysteine-inducible, endoplasmic reticulum stress-inducible, HERPUD1 ubiquitin-like domain member 1 2.571099986 (HERPUD1), transcript variant 1, mRNA [NM_014685] BU733098 UI-E-CI1-afo-h-12-0-UI.s1 UI-E-CI1 Homo sapiens cDNA clone UI- BU733098 2.637830353 E-CI1-afo-h-12-0-UI 3', mRNA sequence [BU733098] Homo sapiens exosome component 5 EXOSC5 2.692247637 (EXOSC5), mRNA [NM_020158] Homo sapiens potassium voltage-gated channel, subfamily G, member 1 KCNG1 2.703684623 (KCNG1), transcript variant 2, mRNA [NM_172318] Homo sapiens SH2 domain protein 2A SH2D2A 2.75980934 (SH2D2A), mRNA [NM_003975] AL581249 AL581249 Homo sapiens B CELLS (RAMOS CELL LINE) Homo AL581249 2.775465209 sapiens cDNA clone CS0DG001YB03 3-PRIME, mRNA sequence [AL581249] THC2269172 Unknown 2.927166352 Homo sapiens histone 2, H2aa3 HIST2H2AA3 2.938759743 (HIST2H2AA3), mRNA [NM_003516] Homo sapiens tyrosyl-tRNA synthetase YARS 2.94243008 (YARS), mRNA [NM_003680] HSU18930 MHC class I antigen HLA- THC2268341 A2 {Homo sapiens;} , partial (24%) 2.965358743 [THC2268341] Homo sapiens solute carrier family 1 (neutral amino acid transporter), SLC1A5 2.99104015 member 5 (SLC1A5), mRNA [NM_005628] Homo sapiens cathelicidin antimicrobial A key anti-microbial CAMP 3.005000572 peptide (CAMP), mRNA [NM_004345] peptide (49).

Homo sapiens Rho GTPase activating ARHGAP9 protein 9 (ARHGAP9), mRNA 3.051559194 It inhibits ERK2 (46). [NM_032496] Homo sapiens Ras homolog enriched in RHEBL1 brain like 1 (RHEBL1), mRNA 3.093509903 [NM_144593] Homo sapiens cDNA clone ENST00000377492 IMAGE:4826036, containing frame-shift 3.132671285 errors. [BC032035] Homo sapiens cystathionase (cystathionine gamma-lyase) (CTH), CTH 3.14257467 transcript variant 1, mRNA [NM_001902] BF689038 602185294T1 NIH_MGC_43 Homo sapiens cDNA clone BF689038 3.20570321 IMAGE:4299791 3', mRNA sequence [BF689038] Homo sapiens asparagine synthetase It is a down-stream ASNS (ASNS), transcript variant 2, mRNA 3.286716977 target of TRIB3-ATF4 [NM_001673] axis (66). Homo sapiens solute carrier family 5 (sodium/glucose cotransporter), SLC5A12 3.304062131 member 12 (SLC5A12), transcript variant 2, mRNA [NM_178498] THC2375394 Unknown 3.3240169 Homo sapiens tribbles homolog 3 Negative regulator of NF-κB (50), it TRIB3 (Drosophila) (TRIB3), mRNA 3.393634165 suppresses adipocyte [NM_021158] differentiation (63). PREDICTED: Homo sapiens LOC645733 hypothetical LOC389025 (LOC389025), 3.397325086 mRNA [XM_374004] Homo sapiens interleukin 29 (interferon, IL29 3.503097918 lambda 1) (IL29), mRNA [NM_172140] Homo sapiens EF-hand calcium binding EFCBP2 protein 2 (EFCBP2), mRNA 3.509125629 [NM_019065] GDF15 Homo sapiens growth differentiation overexpressing mice GDF15 3.593744623 are lean and show factor 15 (GDF15), mRNA [NM_004864] reduced inflammatory responses (94). Homo sapiens potassium channel KCTD15 tetramerisation domain containing 15 3.651733545 (KCTD15), mRNA [NM_024076] THC2340759 Unknown 3.871258178 Homo sapiens phosphoenolpyruvate carboxykinase 2 (mitochondrial) PCK2 (PCK2), nuclear gene encoding 3.88005262 mitochondrial protein, transcript variant 1, mRNA [NM_004563] Homo sapiens stanniocalcin 2 (STC2), STC2 3.951302081 mRNA [NM_003714] Homo sapiens DNA-damage-inducible It is a down-stream DDIT3 transcript 3 (DDIT3), mRNA 6.59893248 target of TRIB3-ATF4 [NM_004083] axis (66).

AJHYNG asparagine synthase (glutamine-hydrolysing) THC2363646 [similarity] - golden hamster 7.040593326 {Mesocricetus auratus;} , partial (17%) [THC2363646]

3. Supplemental Figures Supplementary Figure S1

200

150

100 = 100%) =

Fluorescence

control (

0 AA (μM): 0 0 0 0 50 50 50 50 CBD (nM): 0 1 10 100 0 1 10 100 Supplemental Figure 1. Low concentrations of CBD exert no effect on either basal or AA-induced lipid synthesis of human sebocytes Neutral lipid synthesis (Nile Red staining). Cells were treated by arachidonic acid (AA) and/or cannabidiol (CBD) for 48 hrs. Data are expressed as mean±SEM of four independent determinations as the percentage of the vehicle control (100%, solid line). One additional experiment yielded similar results.

Supplementary Figure S2

A B C

DilC1(5) (↓: apoptosis) SYTOX Green (↑: necrosis) 120 140 120 100 120 100 100 80 80

= 100%) =

= 100%) = = 100%) = intensity 60 60 60

40 40 40

Fluorescence

control

Signal control

( *** ( ( 20 *** 20 *** 20 0 0 0 CBD (µM): 0 50 CBD (µM): 0 50 CBD (µM): 0 50

D E

120 120 100 100 80 80

* *

= 100%) = intensity 60 100%) = 60

40 40

Fluorescence

Signal control

control ( 20 ( 20 0 0 CBD (µM): 0 10 CBD (µM): 0 10

Supplemental Figure 2. High concentrations or long-term applications of CBD decrease human sebocyte viability and lipid production (A) MTT-assay. Viability of sebocytes following 48-hr treatments. CBD: cannabidiol. ***P<0.001. (B) Cell death (DilC1(5)-SYTOX Green double labeling) assays (after 24- hr treatments). (C) Neutral lipid synthesis (Nile Red staining). Cells were treated by CBD for 48 hrs. ***P<0.001. (D) MTT-assay. Viability of sebocytes following 6-day treatments. *P<0.05. (E) Neutral lipid synthesis (Nile Red staining). Cells were treated by CBD for 6 days. *P<0.05. (A, B, C, D, E) Results are expressed in the percentage of the vehicle control (100%, solid line) as mean±SEM of four independent determinations. One additional experiment yielded similar results.

Supplementary Figure S3

200

150

= 100%) = 100

Fluorescence

control ( 0 AA (µM): 50 50 50 50 50 50 CBD (µM): 0 0 0 5 5 5 AM251 (µM): 0 10 0 0 10 0 AM630 (µM): 0 0 10 0 0 10 Supplemental Figure 3. Effects of CBD are not mediated by CB1 or CB2 receptors Neutral lipid synthesis (Nile Red staining). Cells were treated with arachidonic acid (AA), cannabidiol (CBD), AM251 (CB1 receptor antagonist), AM630 (CB2 receptor antagonist) or combinations for 24 hrs. Data are expressed as mean±SEM of four independent determinations as the percentage of the vehicle control (100%, solid line). One additional experiment yielded similar results.

A B

TRPV1 confluence: confluence: 1.5*101,5x10-2 -2 30% 30% 60% 90% pc.

60% -2 1*101,0x10-2 90%

= 1) = expression post confluent TRPV2

-3 confluence: 5*105,0x10-3

mRNA 90% pc.

GAPDH 30% 60%

( (GAPDH = 1)

1,0x10-4 -4

Relative 1*10 Relative mRNA expression 0 0,0 TRPV4 TRPV1 TRPV2 TRPV4 TRPV1 TRPV2 TRPV4 confluence: 30% 60% 90% pc.

TRPV1 TRPV2 NC TRPV4

Supplemental Figure 4. Human SZ95 sebocytes express certain TRP channels (A) Quantitative “real time” PCR on SZ95 sebocyte samples harvested at different confluences. Data of TRPV1, TRPV2 and TRPV4 mRNA expressions were normalized to the level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of the same sample, and are expressed as mean±SEM of three independent determinations. Two additional experiments yielded similar results. (B) Western blot analysis of lysates of SZ95 sebocytes (pc: post-confluent culture). (C) Immunocytochemistry. TRPV1, TRPV2 and TRPV4-specific immunreactivity was determined by immunofluorescence labeling (Alexa-Fluor®-488, green fluorescence) in SZ95 sebocytes. Nuclei were counterstained by DAPI (blue fluorescence). NC: negative control. Scale bars: 20 μm.

120

100

80 = 100%) =

Fluorescence control ( 40

0 HC (µM): 0 1 10

Supplemental Figure 5. Antagonism of TRPV4 exerts no effect on basal lipid synthesis of human sebocytes Neutral lipid synthesis (Nile Red staining). Cells were treated with HC067047 (HC) for 48 hrs. Data are expressed as mean±SEM of four independent determinations as the percentage of the vehicle control (100%, solid line). One additional experiment yielded similar results.

Supplementary Figure S6

A B

2000 2000 control @: +90 mV 1500 I @ +90 mV 1500 GSK @:I @ -90 +90 mV mV 1000 GSK+HC 1500 1000 500

500 ) )

GSK 10 nM pA 0

pA 0 I ( I 1000 ( I HC 10 µM -500 -500 -1000 -1000 -1500 500 -1500 0 50 100 150 200 250 300 350 400 450 500 550 (s) -100 -50 0 50 100 V (mV) 0 10 nM GSK C 10 uM HC I (pA) I D -500 control GSK 100 nM 2+ GSK 100 nM (low [Ca ]EC) 1000 -1000 GSK 100 nM + RR 10 μM GSK 100 nM + HC 1 μM 800

25 RF

= 100%) = 600 -1500 20

0 50 100 150 200 250 300 350 400 450 500 peak RF 15 400

Normalized *** *** 200 ***

10 time (s) control (

5 0 Normalized 0 GSK (nM): 100 100 100 100 2+ low [Ca ]EC: - + - - RR (µM): 0 0 10 0 Time (s): 0 50 100 150 200 250 300 HC (µM): 0 0 0 1 Supplemental Figure 6. TRPV4 is functionally expressed by human sebocytes (A) Time course of whole current at -90 and +90 mV in SZ95 sebocytes treated by 10 nM GSK1016790A (GSK) and 10 µM HC067047 (HC), as indicated in the figure. (B) Current-voltage traces at different time points as indicated in panel A. (C) Fluorescent Ca2+-imaging. Compounds were applied as indicated by the arrow. Fluorescence (measured in relative fluorescence units, RF) was normalized to the 2+ baseline. GSK: GSK1016790A, HC: HC067047, RR: ruthenium red, low [Ca ]EC: nominally Ca2+-free Hank’s solution. (D) Statistical analysis of the Ca2+-imaging data shown in panel C. Measured peak values were expressed in the percentage of the peak value of the control (100%, solid line) as mean±SEM of 3 independent determinations. Two additional experiments yielded similar results. ***P<0.001 compared to the GSK-treated group.

A B 66 200 CBD 5 μM 5 CBD 5 μM + CPZ 5 μM 150

4 RFU

= 100%) = 100 3 44 2

Fluorescence 50

control ( 1 0 Normalized AEA (µM): 30 30 30 0 CBD (µM): 0 5 5 22 0 0 5 CPZ (µM): Time (s): 0 30 60 90 120 150 180

300 00

RF 200 = 100%) =

peak -2-2 100

Normalized 00 30 60 9090 120120 150150 180180

control (

0 CBD (µM): 5 5 CPZ (µM): 0 5 Supplemental Figure 7. Effects of CBD on human sebocytes are not mediated by TRPV1 (A) Neutral lipid synthesis (Nile Red staining). Cells were treated by anandamide (AEA), cannabidiol (CBD), capsazepine (CPZ), or combination for 24 hrs. Data are expressed as mean±SEM of four independent determinations as the percentage of the vehicle control (100%, solid line). Two additional experiments yielded similar results. (B) Fluorescent Ca2+-imaging. Compounds were applied as indicated by the arrow. Fluorescence (measured in relative fluorescence units, RF) was normalized to the baseline (100%). (C) Statistical analysis of the Ca2+-imaging data. Measured peak values were expressed in the percentage of the peak value of the control (100%, solid line) as mean±SEM of 3 independent determinations. Two additional experiments yielded similar results.

Supplementary Figure S8 1.2 A B 1

0.8 expresion

of SCR = = 1) SCR of 0.6 TRPV1 mRNA 0.4 OD: 1 0.85 0.48 0.45

expression 0.2

( β-actin Relative 0

1.2 C D 1

0.8

expresion of SCR = = 1) SCR of

0.6 TRPV2 mRNA 0.4 OD: 1 0.65 0.23 0.31

expression 0.2

( β-actin Relative 0

1.2 E F 1

0.8

expresion of SCR = = 1) SCR of

0.6 TRPV4 mRNA 0.4 OD: 1 0.67 0.04 0.02

expression 0.2 ( Relative β-actin 0

Supplemental Figure 8. Evaluation of efficacy of selective gene silencing of TRPV channels (A, C and E) TRPV1, TRPV2 and TRPV4 mRNA expressions two days after their selective gene silencing. Data are presented by using ΔΔCT method regarding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or peptidil-prolyl isomerase A (PPIA) normalized TRPV mRNA expression of the appropriate scrambled (SCR) control as 1 (solid line). Data are expressed as mean±SD of three independent determinations. (B, D and F) Western blot analyses of lysates of un-transfected (UC), non-sense RNA transfected (SCR: scrambled control), and TRPV1-, TRPV2- and TRPV4-silenced SZ95 sebocytes, respectively, three days after the transfection. OD: β-actin-normalized optical density of the corresponding bands. “siV1a”, “siV1b”, “siV2a”, “siV2b”, “siV4a” and “siV4b” mark different siRNA constructs against TRPV1, TRPV2 and TRPV4, respectively.

Supplementary Figure S9

A B

250 250 200 200 150 150

100 100

(UC = 100%) = (UC

(UC = 100%) = (UC Fluorescence 50 Fluorescence 50 0 0 CBD (µM): 0 5 5 5 CBD (µM): 0 5 5 5 AA (µM): 50 50 50 50 AEA (µM): 30 30 30 30 ‘‘siV2a’’‘‘siV2b’’ SCR ‘‘siV1a’’‘‘siV1b’’ SCR Supplemental Figure 9. Neither TRPV1, nor TRPV2 mediates the lipostatic effects of CBD (A and B) Neutral lipid synthesis (Nile Red staining) following selective gene silencing of TRPV1 (A) or TRPV2 (B) channels (24-hr treatments, started at day 3 after transfection). Data are expressed as mean±SEM of four independent determinations as the percentage of the untransfected vehicle control (100%, solid line). Two additional experiments yielded similar results. SCR: scrambled control. In panel A “siV1a” and “siV1b” mark different siRNA constructs against TRPV1, whereas in panel B “siV2a” and “siV2b” mark two different siRNA constructs against TRPV2. AA: arachidonic acid, AEA: anandamide, UC: untransfected vehicle control.

Supplementary Figure S10

1000

800 RF

= 100%) = 600

peak 400 Normalized 200 ***

control *** (

0 GSK (nM): 100 100 100 SCR ‘‘siV4a’’ ‘‘siV4b’’ Supplemental Figure 10. Silencing of TRPV4 effectively abrogates its functionality Statistical analysis of fluorescent Ca2+-imaging data, obtained after GSK1016790A (GSK) challenges on TRPV4 “silenced” SZ95 sebocytes. Measured peak values were expressed in the percentage of the peak value of the control (100%, solid line) as mean±SEM of 8 independent determinations. One additional experiment yielded similar results. “siV4a” and “siV4b” mark two different siRNA constructs against TRPV4. ***P<0.001.

A B

250 200

200 150 150 100

100

(VC 100%) (VC =

Fluorescence (VC 100%) (VC = Fluorescence 50 50

0 0 AEA (µM): 30 30 30 30 30 30 AA (µM): 50 50 50 CBD (µM): 0 10 10 10 10 10 CBD (µM): 0 10 10 WM (µM): 0 0 0.1 0 0 0 0 0 0.1 Gö (µM): 0 0 0 0.1 0 0 H89 (µM): GF (µM): 0 0 0 0 0.1 0 CSA (µM): 0 0 0 0 0 2 Supplemental Figure 11. Lipostatic effect of CBD is not mediated by activation of PKC, PI3K, calcineurin (PP2B) or PKA Neutral lipid synthesis (Nile Red staining; 24-hr treatments) following various inhibitor treatments in combination with anandamide (A) or arachidonic acid (B). Data are expressed as mean±SEM of four independent determinations as the percentage of the vehicle control (100%, solid line). One additional experiment yielded similar results. AA: arachidonic acid, AEA: anandamide, CBD: cannabidiol, CSA: Cyclosporine A (inhibitor of calcineurin), GF: GF109203X (inhibitor of the conventional and novel PKC isoforms), Gö: Gö6976 (inhibitor of the conventional PKC isoforms), H89 (inhibitor of PKA), PKA: protein kinase A, PKC: protein kinase C, PI3K: phosphatidyl-inositol-3-kinase, PP2B: protein phosphatase 2B, WM: wortmannin (inhibitor of phosphatidyl-inositol-3-kinase).

Supplemental Figure 12. Hierarchy of down-regulated Biological Networks Gene Ontology (BiNGO) terms following CBD treatment (10 μM, 24 hrs). Size of the nodes is proportional to the number of the identified genes of the corresponding GO term, whereas color of each node correlates to the adjusted P-value of the GO term (bigger size and more yellow color indicate higher number and significance, respectively)

intracellular organelle part intracellular

cytoplasm cytoskeleton cytoskeletal part intracellular organelle intracellular part intracellular non-membrane-bounded organelle membrane positive positive regulation of non-membrane-bounded cytoplasmic regulation of organelle part cGMP cGMP organelle part metabolic cell part biosynthetic cell process process actin organelle cytoskeleton membrane part plasma phosphorylase podosome membrane kinase complex regulation of regulation of positive cellular_component positive cGMP cGMP regulation of regulation of biosynthetic metabolic cyclic cyclic process process laminin-2 nucleotide nucleotide Toll-like complex biosynthetic plasma metabolic receptor process protein complex membrane part process 1-Toll-like receptor 2 macromolecular protein arylamine extracellular phosphorylase serine complex N-acetyltransferase region kinase activity complex activity C-palmitoyltransferase activity regulation of regulation of cyclic cyclic laminin cell junction nucleotide nucleotide extracellular biosynthetic complex metabolic region part positive process positive process laminin-8 regulation of regulation of negative complex regulation of nucleotide nucleotide survival gene biosynthetic metabolic N-acetyltransferase process process calmodulin-dependent C-palmitoyltransferase product extracellular activity anti-apoptosis positive protein kinase activity expression regulation of matrix part activity positive DNA strand laminin-10 regulation of elongation basal lamina complex anchoring positive negative regulation of positive DNA extracellular junction regulation of regulation of protein amino positiveregulation of matrix regulation of replication regulation of regulationinterleukin-6 of apoptosis DNA acid nucleotide nucleotide tumor necrosisbiosynthetic replication ADP-ribosylation biosynthetic metabolic acetyltransferase factor process regulation of negative process process activity negative biosynthetic survival gene regulation of positivenegative regulation of process product positivegene basement C-acyltransferase regulationregulation of of protein palmitoyltransferase regulationexpression of positive N-acyltransferase activity apoptosis expression positive membrane activity positive cellular nucleobase,macromolecule positive regulation of serine/threonine activity regulation of regulation of nucleoside,biosynthetic negative regulation of regulation of kinase activity biosynthetic regulation of telomerenegative macromolecule processregulation of DNA metabolic DNA strand proteinaceous adherens cytokine process nucleotide and maintenanceregulation of biosynthetic cellular DNA extracellular junction biosynthetic nucleic acid process vianucleobase, telomerase elongation replication matrix process process metabolic biosynthetic negative negative nucleoside, regulation of process process regulation of regulation of regulation of nucleotide and protein thiopurine DNA metabolic telomere tumor necrosispositive nucleic acid modification S-methyltransferase dolichyl-phosphate process maintenance factorregulation of metabolic process activity acyltransferase beta-D-mannosyltransferase negative regulation of regulation of via telomerase biosyntheticprogrammed process activity activity regulation of apoptosis interleukin-6 processcell death regulation of regulation of biosynthetic transforming cellular negative nucleobase, process positive growth negative regulation of biosynthetic regulation of nucleoside, regulation of regulation of regulation of protein kinase factor-beta3 regulation of gene process macromoleculenucleotide and regulation of macromolecule macromolecule DNA metabolic activity production programmed expression positive metabolic nucleic acid telomere positive biosynthetic metabolic process cell death positive process maintenance regulation of regulationregulation of of processregulation of processnegative metabolic regulation of negative viaregulation telomerase of spindle cytokinebiosynthetic cellular regulation of process nitrogen metabolic regulation of positive cellular protein organization S-methyltransferase biosyntheticprocess biosynthetic regulation of compound process cellular regulation of metabolic activity negative process process regulation of mannosyltransferase metabolic metabolic proteintelomere negativeprocess S-adenosylmethionine-dependent transferase regulation of metabolic regulation of mitotic spindle activity, activity regulation of process process maintenance negative methyltransferaseG-protein transforming process nitrogen organization transferring transforming regulation of activitycoupled prenylcysteine growth regulation of negative positive compound acyl groups growth positive telomere receptor phosphotransferase oxidase activity factor-beta regulationprogrammed of regulation of regulation of metabolic G-protein other than factor-beta3 regulation of regulation of maintenance activity activity, alcohol production tumor necrosiscell death cell death organelle process regulation of coupled amino-acyl production cell death macromoleculeregulation of group as factor organization microtubule photoreceptor groups metabolic cellular negative acceptor regulationproduction of cytoskeleton activity glucan regulation of process metabolic regulation of regulation of regulation of kinase activity interleukin-6 organization 1,4-alpha-glucosidase biosynthetic process organelle chromosome cell cycle production regulation of activity positive regulation of positive positiveprocess organization organization process negative nitrogen regulation of regulation of transforming regulation of regulation of regulation of regulation of transferase negative positive regulationcompound of primary estrogen growth cell metabolicregulation of telomere mitotic cell methyltransferase activity, regulation of negativemetabolic metabolic metabolic regulation of receptor factor-beta communication process cellular maintenance cycle activity transferring cytokine regulation ofprocess process process cytoskeleton oxidoreductase signaling production process hexosyl groups production regulation of cellular Wnt receptor organization activity, acting pathway regulation of alkylbase DNA cytokine process signaling transmembrane on sulfur group positive cell death transferase N-glycosylase production positive regulation of pathway, receptor of donors, regulation of transferase activity, activity positive regulation of organelle non-canonical oxygen as steroid planar cell activity activity, transferring regulation of homeostatic organizationregulation ofregulation of Wnt receptor acceptor hormone polarity photoreceptor transferring acyl groups regulation of cellular process microtubule-basedcell cycle signaling receptor regulation of pathway activity phosphorus-containing positive regulation of negative component negativeprocess pathway glucosidase cell groups transferase regulation of estrogen signaling regulation of organizationmetabolic regulation of activity regulation of communication regulation of transferase activity, telomerase receptor pathway multicellular process negativehomeostatic branching cellular activity, transferring activity signaling positive organismal process regulation ofprocess involved in regulation of transferring glycosyl pathway process cellular prostate gland regulation of one-carbon groups positive establishment component morphogenesis signaling groups regulation of negativeof planar organization regulation of pathway oxidoreductase positive regulation of biological regulation polarityof steroid regulation of regulation of regulation of activity, acting DNA regulation of anatomical process biological transferase hormone morphogenesis multicellular homeostatic on sulfur group N-glycosylase transferase structure process activity receptor of a branching organismal process receptor activity of donors hydrolase activitysingle-stranded activity morphogenesis regulation of single-stranded signaling structure process Wnt receptor activity, DNA binding positive regulation of cellular telomeric DNA pathway signaling hydrolyzing regulation of telomerase component pathway binding regulation of O-glycosyl helicase activity organization signaling compounds negativeactivity regulation of regulation of regulation of pathway biological female gamete positive positive developmental NEDD8 telomerase process generation regulation of regulation of process activating oxidoreductase activity hydrolase regulation of enzyme activity activity hydrolase catalytic regulation of activity transferase activity, activity branching activity hydrolyzing negative involved in small protein N-glycosyl regulation of salivary gland venous blood activating hydrolase positive signal compounds transferase morphogenesis vessel enzyme activity activity, acting regulation of regulation of estrous cycleovulation transducer structure-specific activity gamete morphogenesis negative regulation of phase activity catalytic activity on glycosyl helicase regulation of molecular generation angiogenesiscell surface telomerase DNA binding regulation of catalytic bonds telomeric DNA activity hydrolase function receptor linked inhibitor activity catalytic development of binding activity activity negative activity ovarian folliclesignaling ovulation from regulation of regulation of primary female rupture pathwayovarian follicle molecular molecular blood vessel sexual regulation of enzyme function function morphogenesis characteristics venous blood branching two-component hydrolase inhibitor activity lymphangiogenesisfemale sex vessel involvedregulation in of biological response molecular activity sexual signaling development salivary glandbiological regulation differentiation female gonad regulator reproduction pathway morphogenesis transducer morphogenesisquality development activity of a branching activity enzyme neurological by extracellular reproductive nitric oxide multicellularovulation cycle DNA binding gas matrix-epithelial process in a epithelium regulator homeostasis system organism homeostasis chemical cell signaling activity myeloid leukocyte process multicellularovulation cycle vasculature homeostasis cognition system process reproduction sex tissue nucleic acid sequence-specific leukocyte mediated organismanatomical process developmentbranching transcription immune differentiation organ morphogenesisblood vessel binding DNA binding mediated immunity structure involved in regulator molecular_function effector morphogenesis morphogenesis development mast cell immunity homeostatic formation development of prostate gland activity response to process of a branching lymph vessel sensory process involved in primary sexual morphogenesis mediated temperature structure ovarian follicle G-proteindevelopment rhythmic morphogenesis anatomical characteristics immunityperception of stimulus development coupled gonad sensory signalingprocess multicellular structure prostate temperature response to immune receptor developmentmorphogenesis binding extracellular stimulus perception extracellularsystem organismal morphogenesis prostate gland of an epithelial cord detection of abiotic stimulus multicellular protein matrix bindinghydrolase immune matrix-cellprocess reproduction process stromal signaling epithelium arborization hydrolase detection of abiotic stimulus organismal gland activity, acting response signaling morphogenesis pathway organ involved in activity, acting detection of stimulus behavior development on acid detection of reproductive morphogenesis prostate gland prostate on acid temperature involved in multi-organism development anhydrides, in pyrophosphatase temperature learning or process epithelium glandular anhydrides stimulus sensory response to process developmental phosphorus-containing activity stimulus detection of memory reproductive acinus perception cellular biological_process morphogenesis anhydrides involved in temperature external process follicle-stimulating component developmentalanatomical morphogenesis sensory stimulus stimulus hormone prostate detection ofresponse to response to biogenesis cell-cell process structure reproductive transporter laminin binding perception of involved in prostate gland tissue glandular sensory stimulusendogenous stimulus signaling signaling development structure activity pain sensory detection of localization morphogenesis development acinus perception of stimulus cellular pathway development gland external response to morphogenesis nucleoside-triphosphatase pain perception cellular component establishment development laminin-1 other organism developmental system activity stimulusresponse to organization of localization protein binding binding memory maturation developmentprostate response toendogenous response to glandular hydrolase hormone stimulus chemical epithelial-mesenchymal acinus activity, acting response to urogenital stimulus detection of stimulus cell development on acid response to reproductive cell signaling systemepithelium chemical stress communication anhydrides, enzyme binding steroid anatomical response to cellular development stimulus development catalyzing hormone structure biotic stimulus membranemacromolecule process response to cellular epithelial cell epithelial cell transmembrane stimulus homeostasisdetection of organization localization establishment estrogen component maturation differentiation substrate-specific movement of short-term biotic stimulus of protein prostate gland stimulus assembly metabolic cellular process cellular involved in transporter DEAD/H-box cellular establishment localization involved in development nervous transmembrane substances memory response to developmental activity RNA helicase response to process of localization epithelial-mesenchymalprostate glandprostate gland system transporter detection of organicresponse to process binding stimulusnitrogen organelle in cell developmentsignaling development secretory development activity bacterium substancebacterium cell ATPase ATPase activity compound organization involved in columnal cellular transport development activity, cellular metabolic cell projection cellular prostate gland luminar neurotransmitter response to epithelial cell coupled to response to process organization localization development epithelial cell transporter receptor hormone differentiation transmembrane ATPase chemical cell cycle differentiation activity binding cellular detectionstimulus of response to movement of activity, stimulus cellular involved in response to molecule of molecule of glandular substances coupled to cellular cellular membrane cell organic acid prostate steroid bacterial origin bacterial origin epithelial cell movement of response to response to cell maturation transportepithelial cell glandular hormone chromosome organization differentiation differentiation substances estrogen telomere biotic stimulus cellular maturation acinus active stimulus organizationsmall molecule primary primary active ATPase stimulus assembly response to macromolecule development transmembrane P-P-bond-hydrolysis-driven G-protein-coupled detection of metabolic metabolic protein transmembrane activity, cellular stress metaboliccatabolic protein transporter transmembrane receptor bacterial process process localization transporter response to process process transport neuronneurogenesis columnar/cuboidal activitysubstrate-specific transporter binding coupled lipoprotein cellular epithelial cell activity organic development epithelial cell transmembrane activity cell projection metabolic development substance cellular intracellular differentiation transporter assembly process response to macromolecule transport activity alcohol ATPase bacterial localization amine transport organic acid telomere metabolic activity, lipoprotein cellular telomere process microvillus transmembrane maintenance cellular secondary hormonecoupled to response to organization organization mitotic cell transporter nitrogen carboxylic acid neurotransmitter:sodium active receptortransmembrane molecule of nucleobase, cycle activity detection of sulfur compound neuron symporter transmembrane movement of nucleoside, cellular lipid cellular amino lipid metabolic transport binding bacterial bacterial origin macromolecule metabolic differentiation activity ions nucleotide and metabolicmetabolic process transporter lipopeptide modification processacid and cellular neuron generation of ion frizzled binding nucleic acid processprocess protein activity derivative aromatic maturation neurons transmembrane metabolic metabolic transport within ATPase telomere response to compound amine transporter processsmall molecule process lipid bilayer intracellular activity, response to capping DNA damage cellular metabolic norephinephrine:sodium transmembrane activity telomere catabolic protein protein coupled to bacterial stimulus macromolecule macromoleculeprocess heterocycle symporter transporter formation via process metabolic transport transmembrane lipopeptide nucleobase, metabolic catabolic cellular metabolic activity activity cellular telomerase process movement of nucleoside and process process catabolic process basic amino amino acid ions, response to cellular protein acid transport prenol nucleotide process transport bacterial localization phosphorylative metabolic metabolic symporter anion detection of lipoprotein nucleic acid DNA metabolic carboxylic acid monoamine mechanism process process chemokine activity transmembrane triacylated metabolic process neuron transmembranetransmembrane isoprenoid receptor transporter bacterial process microvillus remodeling histidine transporter transporter metabolic endomitotic transport within nuclear activity lipopeptide assembly B cell receptor transport activity activity response to process cell cycle lipid bilayer solute:sodium hormone cellular transport within triacylated nucleobase, symporter receptor nitrogen protein lipid bilayer cation bacterial nucleoside, activity binding compound transport into norepinephrine transmembrane lipopeptide nucleoside nucleotide S-adenosylmethionineand cellular amino cellular protein protein chloride-transporting catabolic protein transmembrane transporter cellular metabolic metabolic acid derivative metabolic membrane raft ATPase DNA nucleic acid processcellular amino transport out of transporter activity response to process catabolic heterocyclecatabolic activity DNA repair process modification catabolic acid derivativeprocess membrane raft activity amino acid solute:cation bacterial processcellular catabolicprocess neutral amino process metabolic transmembrane symporter lipopeptide nucleobase, macromolecule process proteolysis acid transport process transporter activity nucleoside and catabolic inorganic anion activity nucleotide process purine L-amino acid transmembrane polyprenol catabolic nucleoside nucleotide transport transporter metabolic glutamine process phosphate metabolic activity process metabolic process B cell receptor transport metal ion process transport into cellular DNA catabolic chemokine transmembrane membrane raft steroid response to process receptor sodium ion transporter chloride purine hormone triacylated transport out of neutral amino transmembrane activity transmembrane nucleoside basic amino receptor bacterial membrane raft acid transporter inorganic transporter metabolic cellular protein acid binding lipopeptide transmembrane activity cation activity process purine catabolic alanine transmembrane ribonucleoside transporter transmembrane ribonucleoside process transport transporter metabolic activity L-amino acid transporter base-excision metabolic purine proteolysis activity prenylcysteine transmembrane activity process process prenylcysteine asparagine repair metabolicnucleotide involved in transporter catabolic modification-dependentnucleotide transport depurination catabolic cellular protein activity process macromolecule process purine nucleotide metabolic process catabolic catabolic deoxyribonucleotide L-alanine catabolic process process monovalent dolichol process metabolic transport base-excision process alanine inorganic metabolic process repair, AP site transmembrane glucocorticoidcation process L-glutamine formation transporter receptortransmembrane transmembrane activity bindingtransporter transporter L-histidine activity activity L-asparagine transmembrane purine transmembrane transporter deoxyribonucleotide transporter modification-dependent L-alanine activity catabolic protein activity process transmembrane catabolic 2'-deoxyribonucleotide transporter deoxyribonucleotideprocess metabolic activity metabolic process deoxyribonucleotide process catabolic process

prenylated protein catabolic process Supplemental Figure 13. Hierarchy of up-regulated Biological Networks Gene Ontology terms following CBD treatment (10 μM, 24 hrs). Size of the nodes is proportional to the number of the identified genes of the corresponding GO term, whereas color of each node correlates to the adjusted P-value of the GO term (bigger size and more yellow color indicate higher number and significance, respectively)

cellular response to insulin stimulus

cellular cellular response to response to response to cellular glucocorticoid interleukin-1 response to response tointerleukin-6 stimulus tumor necrosis response to insulin stimulus protein-pyridoxal-5-phosphate response to factor tumor necrosis linkage response toresponse to interleukin-6 factor follicle-stimulatinginterleukin-1 hormone protein-pyridoxal-5-phosphate stimulus response to cellular linkage via corticosteroid response to peptidyl-N6-pyridoxal stimulus peptide protein-cofactor phosphate-L-lysine response to hormone linkage amphetamine response to cellular stimulus response to methotrexate apoptosis in cytokine response to peptidyl-lysine response tostimulus modification response to endoplasmic peptide reticulum response tocytokine hormone cellular stress response to gonadotropinstimulus stimulus response to cellular steroid stimulus lipopolysaccharideresponse to defense hormone response to peptidoglycan response to stimulus organic cyclic response to post-translational Gram-negative cellular mRNA response to substance lipopolysaccharide response to protein bacterium response to peptidyl-amino amine stimulus peptidoglycan modification transcription hormone acid from RNA complement cellular stimulus modification polymerase II receptor response to response to promoter mediated cellular protein glycerol amino acid signaling response to stimulus biosynthetic stimulus cellular pathway glucoseresponse to response to process mRNA starvationprotein response to organic fermentation L-fucose stimulus hormone substance apoptosis transcription response to glycerol metabolic stimulus transcription organic response to cellular metabolic process defense homophilic cellheterophilic nutrient levels response to process from RNA substance adhesion cell-cell response to molecule of polymerase II fucose bacterium adhesion promoter cellular bacterial origin protein metabolic immune response to fucose endoplasmic response to response to macromolecule modification process response-activating organic catabolic reticulum molecule of endogenous glycosylation process L-fucose cell surface nitrogen process unfolded response to bacterial origin stimulus hexose catabolic receptor protein acid catabolic process signaling response cellular glycoproteinalditol process pathway response to regulation of cellular biosyntheticbiosynthetic tyrosyl-tRNA response to unfolded process aminoacylation granulocyte response to protein amino process transcription, starvation protein glycosylation chemotaxis immune response to nutrient levels DNA-dependent cellular energy acid polyol response-regulating responsebacterium to cellular derivationglycosylation by response to alditol biosynthetic cell surface defense ER overload response to starvation oxidation of metabolic process receptor response unfolded chemical positive response to response response to macromolecule organic process hexose glucoseseryl-tRNA signaling protein stimulus metabolic regulation of regulation of nervous endoplasmic response to extracellular modification compounds transcription metabolic aminoacylation pathway endogenous cellular cellular process regulation ofmacrophage MHC class I systemER-nucleus reticulum cellular stimulus monosaccharide process response to programmed carbohydrate polyol chemotaxis biosynthetic development stress stimulus response to cellular protein positive neutrophil regulation of signaling chemical response to cell death catabolic catabolic process external glycoprotein metabolic regulation of chemotaxis mononuclear pathway stimulus biotic stimulus cell-cell metabolic process regulation of stimulus processmetabolic process tryptophanyl-tRNA MHC classregulation I of cell migration immune cell surface adhesion process translation tRNA hexose vascular gene process aminoacylation biosyntheticmacrophage response-activating receptor linked aminoacylation biosynthetic positive endothelial expression process chemotaxis signal signaling for protein gluconeogenesis system response to process regulation regulationgrowth of factor transduction translation positive pathway development monosaccharide vascular leukocyteproduction biotic stimulus macromolecule cellular monosaccharide carbohydrate cellular metabolic regulation of endothelial chemotaxis cellular biosynthetic biosynthetic response to biosynthetic carbohydrate process ncRNA neutrophil growth factor response to cellular process macromolecule tRNA external response to biosynthetic processmetabolic chemotaxis positive positive stress response to process cellular biosynthetic metabolic production stimulus extracellular carbohydrate process RNA regulation of regulation of stress carbohydrate process process process positive positive regulation of regulation of immune stimulus catabolic biosynthetic macromolecule behavior metabolic RNA metabolic regulation of regulation of response-regulating cellular process biosynthetic macromolecule gene process carbohydrate process process alcohol leukocyte regulation of signaling response to cellular process response tobiosynthetic expression regulation of regulation of metabolic biosynthetic chemotaxis lipid response to positive external process response to transcriptionpathway in stimulus macromolecule processmacromolecule process biosynthetic other organism generation of pyruvate regulation of stimulus external response neurogenesisto response to metabolic metabolic nucleic acid tRNA process regulation of precursor protein metabolic cytokine stimulusregulation of stress stimulus process process alcohol metabolic aminoacylation positive leukocyteregulation of anatomical metabolitesmetabolic process production behavior metabolic process regulation of migrationchemotaxis regulation of structure and energy process regulation of cell cell death process alcohol negative chemotaxis cytokine regulation of positive development fatty acid differentiation catabolic regulation of positive production immune regulation of activation of cellular biosynthetic signaling cell adhesion process lipid regulation of response immune intracellular immune biosynthetic process positive leukocyte regulation of generation of response signaling response pathway multicellular biosynthetic process biosynthetic cellular monocarboxylic regulation of migration biosynthetic neuronsregulation of pathway process organic acid negative process regulation of regulation of organismal developmental nucleobase, transcription cellular small molecule hydrogen acid metabolic regulation of lipid metabolicbiosynthetic process nitrogen development nucleoside, biosynthetic oxaloacetate regulation of process metabolic biosynthetic antibiotic process process process compoundpositive nucleotide and process sulfide metabolic fatty acid macromolecule process biosynthetic glutamine positive metabolicregulation of death process biosynthetic process biosynthetic metabolicregulation of cellular process nucleic acid amino acid metabolic regulation ofnegative response to immunedevelopmental primary cellular ketone process sulfur process process negative processpositive metabolic carboxylicactivation acid process regulation of regulation of processnucleobase, process metabolic metabolic compound regulation of regulationmacromolecule of regulationstimulus of system cell metabolic processorganic acid biosynthetic cellular nucleoside, regulation of biological process process biosynthetic metabolic immune regulation of process communication process metabolic biosynthetic fatty acid cellular biosynthetic nucleotide andcell migration adhesion process arginine process process system immuneregulation of processcellular amino processoxoacid metabolic biosynthetic process nucleic acid positive signalingbiological_process catabolic metabolic process systemresponse to acid and metabolic carboxylic acid process process metabolic regulation of process small moleculecellular cellular dicarboxylicprocess positive regulation ofpositive regulationprocess of stimulus derivative process metabolic arginine positive regulation of regulationprocess ofmulticellular metaboliccatabolic nitrogen acid metabolic regulation of primary locomotion multicellular cellular metabolic process biosynthetic regulation of protein locomotionorganismal multi-organism processprocess sulfur compound process cell migration metabolic organismal drug metabolic nitrogen process glutamine processfumarate protein negativemetabolic process process metabolic biosynthetic glutamine process regulation of regulation of signaling process process compound family amino metabolic regulation processof process process family amino metabolic metabolic multicellular process pyruvate family process lipid metabolic positive metabolicsmall molecule organic acid cellular amino acid process process organismal amino acidacid metabolic negative process regulation of regulation of regulation of processcatabolic catabolic acid metabolic biosynthetic process cell-cell metabolic process regulation of cellular ketone metabolic localization biological cell killinglocalization cellular nitrogen process process process process positive intracellular signaling sulfur amino process alanine 2-oxoglutarate fatty acid metabolic process negative regulation homeostasis compound hydrogencellular amine regulation of signal acid cellular amino metabolic metabolic metabolic process regulation of regulation of regulation of metabolic metabolic sulfide biosynthetic process process metabolic cellular biological biological transduction process antibiotic process acid process negative regulation of regulation of metabolic carboxylicprocess acid process component process process signal metabolic biosynthetic regulation of cellular angiogenesis process sulfur amino catabolic movement regulation of transmission establishment process process regulation of transmission of process glutamate cellular regulation ofmetabolic regulation of transcription of localization acid metabolic regulation of transcription nerve impulse metabolic metabolic neurogenesisprocess nervous factor activityregulation of process positive cellular protein positive positive regulator amine aspartate process process system cellular signal system process urea metabolic L-alanine glutamate regulation of metabolic positive regulation of regulation of negative activity cellular amide biosynthetic family amino component transduction interspecies catabolic cellular protein development regulation of regulation of regulation of killing of cells process metabolic process metabolic process regulationpositive of cellular cellular amine acid process to organization molecular interactionof another serine familyaspartate process metabolic regulationangiogenesis of component component cellular biological regulation of metabolic process glutaminebiosynthetic positive between amide 2-oxoglutarate positive regulation of process regulation of process process function regulation of organism amino acidfamily amino familyprocess amino cellular movement organization regulation of multi-organism organisms process regulation of phosphate regulation of biological biosynthetic cellularmetabolic amino acid metabolic regulation of phosphorusmetabolic developmental regulationprocess of homeostatic acid catabolic aspartate phosphate metabolic developmentalnegative regulation of quality process acid catabolicprocess process positive neuron metabolic process positive regulation of process signaling process serine familyprocess biosynthetic metabolic process processregulation of signaling cellular process glutamate regulation of differentiation process regulation of anatomical process amino acid process process cellular chemical phosphorus cellular structure pathway biosynthetic catabolic process regulation of homeostasis metabolic process positivemorphogenesis neurological process process positive growth synaptic process regulationregulation of of system regulation of transmission regulation of cell projectiongrowth cysteine cysteine aspartate protein positive process protein regulation of organization urea cycle amine metabolic biosynthetic metabolic asparagine modification regulation of regulation of cell homocysteineprocess process modification neurogenesis positive catabolic process biosynthetic process positive regulation of cell regulation of negative metabolic process development negative symbiosis, transport process process positive regulation of regulation of cell morphogenesis cell positive process regulation of regulation of encompassing asparagine cell projection differentiation negativecommunication regulation of modification of regulation of phosphorylation cell molecular regulation of mutualism organization negative transcription morphology or aspartate metabolic glutamate phosphorylation differentiation regulation of regulation of function molecular regulation of regulator through family amino process catabolic regulation of developmental regulation of DNA binding functionphysiology of positive cell growth activity parasitism interaction with acid catabolic L-serine process to neuron process multi-organism regulation of regulation of other organism killing of cells L-serine regulation of positive regulation of symbiont process biosynthetic aspartate projection negativeprocess symbiosis, binding involved in in other chemical metabolic cell regulation of regulation of anatomical process development regulation of encompassing symbiotic organism homeostasis cellular ion process development developmental developmental positive regulationgrowth of structure size interaction involved in homeostasis regulation of positive mutualism ion transport growthpositive regulation of negativegrowth signal symbiotic positive protein amino regulation of regulation of through positive regulation of cell regulation of transduction positive interaction specific granule regulation of acid cell signaling parasitism regulation of cell negativegrowthcommunication transcription regulation of aspartate protein amino morphogenesis pathway modulation of TOR signaling phosphorylation regulation of factor activity signaling catabolic axonogenesis involved in growth of cascade acid cell regulation of organic acid melanosome differentiation processsymbiont process phosphorylation differentiation intracellular nerve-nerve transport protein kinase involved in synaptic cascade interaction with transmission host regulation of disruptionmodification of by positive cells of otherhost of regulation of axon extensionregulation of negative amine transport regulation of organismsymbiont ion axonogenesis fat cell positive regulation of positive cation transport stored axon extension involvedmorphology in or homeostasis cellular cation differentiation regulation of binding regulation of secretory negative symbioticphysiology homeostasis regulation of signal binding granule regulation of interaction peptidyl-tyrosine transduction negative regulation of growth of phosphorylation regulation of regulation of regulation of lipid pigment positive JAK-STAT symbiont killing by homeostasishost regulation of negative positive DNA binding cellular protein binding granule regulation of cascade involved in of symbiont tyrosine regulation of regulation of component peptidyl-tyrosine interaction with cells metal ion phosphorylation fat cell negativeintracellular size carboxylic acid phosphorylation host transport of STAT differentiation positive regulationprotein of kinase transport protein regulation of CREBcascade modulation of monovalent cell size transcription growth of inorganic cellular di-, cytoplasmic positive factor activity symbiont on or cation tri-valent membrane-bounded synaptic dicarboxylic regulation of positive near host cation transport inorganic membrane-bounded vesicle transmission, acid transport vesicle tyrosine regulation of regulation of disruption by homeostasis cation cell size glutamatergic phosphorylation JAK-STAT host of amino acid homeostasis of STAT cascade symbiont cells transport protein positive acetyl-CoA:L-glutamate3'-5'-exoribonuclease negative aromatic amino regulation of potassium ion cytoplasmic N-acetyltransferase activity regulation of acid transport transport vesicle activity growth of protein binding monocarboxylic fatty acid symbiont on or acid transport homeostasis intracellular near host di-, tri-valent membrane-bounded surface inorganic organelle exonuclease L-amino acid cation sulfur amino cytoplasmic membrane-bounded activity, active transport homeostasis cytoplasm branched-chain acid transport part organelle exoribonuclease with either ribo- tryptophan interleukin-8 aliphatic amino vesicle activity, or amino acid transport producing deoxyribonucleicreceptor import acid transport acids andbinding 5'-phosphomonoesters 3'-5' neutral amino exonuclease producing intracellular acid transport L-alanine NLS-dependent N-acetyltransferase activity5'-phosphomonoesters organelle activity transport protein nuclear CXCR import complex hydroxyproline chemokine transport receptor L-alanine:2-oxoglutarate binding alanine intracellular aminotransferase L-cystine intracellular part transport organelle activity transport galactoside exonuclease 2-alpha-L-fucosyltransferase chemokine acetyltransferase activity activity exoribonuclease receptor activity activity binding leucine L-serine nucleocytoplasmic alanine-oxo-acid transport transport serine transport shuttling transaminase cell fraction leucine import threonine complex activity G-protein-coupled complement proline transport N-acyltransferase component receptor transport cellular_component activity C3a binding exosome interleukin-28 binding soluble fraction receptor (RNase cell part complex) ribonuclease nuclease binding cytokine activity activity receptor alpha(1,2)-fucosyltransferase binding complement activity L-aspartate:2-oxoglutarate component transaminase cell acyltransferase aminotransferase C3a receptor membrane activity activity receptor complement activity complement activity ATP-binding binding receptor binding cassette (ABC) activity protein complex pyridoxal transporter calcium:sodium hydrolase phosphate complex antiporter macromolecular activity complex activity, acting binding calcium:cation transferase on ester bonds transferase solute:solute antiporter activity, plasma activity, protein binding antiporter activity transferring cation:cation membrane membrane part fucosyltransferase transferring activity acyl groups antiporter activity nitrogenous transmembrane other than activity receptor groups receptor G-protein amino-acyl cofactor binding solute:cation complex sialyltransferase activity coupled tyrosine-tRNA groups vitamin B6 antiporter antiporter activity transferase receptor ligase activity binding activity activity ion channel activity, activity hydrolase intrinsic to complex (alpha-N-acetylneuraminyl-2,3-beta-galactosyl-1,3)-N-acetyl-galactosaminide transferring C3a transferase transferase activity inorganic membrane 6-alpha-sialyltransferase glycosyl anaphylatoxin activity, activity, cation activity groups receptor transferring transferring receptor activity transmembrane interleukin-28 activity plasma hexosyl groups acyl groups anaphylatoxin transporter monovalent receptor serine-tRNA vitamin binding transferase binding receptor inorganic complex membrane part ligase activity peptide cation activity activity activity symporter cation cation channel receptor secondary transmembrane activity metal ion transmembrane complex activity, active transporter intrinsic to transmembrane transporter integral to signal G-protein transmembrane activity plasma aminoacyl-tRNA transporter activity membrane transducer peptide coupled transporter membrane ligase activity catalytic activity anion activity transferase activity receptor activity transferase peptide binding transmembrane tryptophan-tRNA activity, activity solute:cation activity, ion transporter ligase activity kinase activity transferring symporter transferring transmembrane activity phosphorus-containing molecular_function activity voltage-gated alkyl or aryl groups transporter potassium potassium ligase activity, integral to potassium (other than oxidoreductase activity sodiumchannel ion activity channel forming active plasma channel methyl) groups activity molecular transmembrane complex aminoacyl-tRNA ligase activity transmembrane cation channel complex ligase activity, transducer transporter membrane acetylglutamate transportersubstrate-specific activity and related forming activity kinase activity activity transmembrane transmembrane compounds carbon-oxygen activity transporter transporter transporter anion:cation bonds activity voltage-gated phosphotransferase activity activity ion channel symporter substrate-specific solute:sodium potassium activity, lyase activity substrate-specific activity activity channel activity symporter channel activity carboxyl group prenyltransferase enzyme transporter activity oxidoreductase as acceptor activity regulator activity organic acid organic activity, acting activity transmembrane acid:sodium on CH-OH voltage-gated transporter symporter group of cation channel ligase activity, activity activity activity donors amine forming transmembrane L-serine carbon-nitrogen voltage-gated carbon-nitrogen carbon-carbon transporter ammonia-lyase bonds passive sodium:dicarboxylateion channel lyase activity lyase activity activity activity ammonia-lyase transmembrane symporteractivity carbon-sulfur carboxylic acid activity activityoxidoreductase transporter lyase activity ligase regulator transmembrane activity, acting activity dicarboxylic phosphoglycerate activity channel activity transporter on the CH-OH acid dehydrogenase activity group of transmembrane activity donors, NAD transporter or NADP as carbon-nitrogen amino acid activity sulfur amino acceptor ligase activity, transmembranegated channel voltage-gated acid with glutamine transporter activity channel activity carboxy-lyase transmembrane as activity L-threonine cystathionine activity transporter amido-N-donor cystathionine ubiquitin-protein ammonia-lyase activity beta-lyase gamma-lyase ligase regulator activity activity activity activity L-alanine L-cystine L-amino acid transmembrane transmembrane asparagine phosphoenolpyruvate neutral amino transmembrane transporter transporter synthase carboxykinase acid transporter activity (glutamine-hydrolyzing) activity transmembrane activity phosphatidylserine activity activity decarboxylase transporter alanine phosphoenolpyruvate activity transmembrane activity L-threonine carboxykinase serine transporter transmembrane (GTP) activity transmembrane activity transporter transporter L-proline activity activity transmembrane L-serine L-hydroxyprolinetransporter transmembrane transmembrane activity transporter transporter activity activity Supplementary Figure S14

A B

1.2 NRIP1

1 = = 1)

0.8 OD: 1 0.81 0.17 0.10

of SCR SCR of 0.6

0.4 β-actin

0.2 (expression

Relative mRNA expressionmRNARelative 0

Supplemental Figure 14. Evaluation of efficacy of selective gene silencing of NRIP1 (A) NRIP1 mRNA expression two days after selective gene silencing. Data are presented by using ΔΔCT method regarding peptidyl-prolyl isomerase A (PPIA) normalized NRIP1 mRNA expression of the scrambled (SCR) control as 1 (solid line). Data are expressed as mean±SD of three independent determinations. (B) Western blot analysis of lysates of untransfected (UC), non-sense RNA transfected (SCR: scrambled control), and NRIP1-silenced SZ95 sebocytes three days after the transfection. OD: β-actin-normalized optical density of the corresponding bands. “siNRIP1a” and “siNRIP1b” mark two different constructs against NRIP1. NRIP1: nuclear receptor interacting protein-1.

Supplementary Figure S15

A B

1.2 TRIB3

1 = = 1) 0.8 OD: 1 0.51 0.24 0.19 of SCR SCR of 0.6

0.4 β-actin

0.2 (expression

Relative mRNA expressionmRNARelative 0

C 200 150 100

Fluorescence 50 (SCR = 100%) = (SCR 0 CBD (µM): 0 10 10 10 AA (µM): 50 50 50 50

Supplemental Figure 15. Evaluation of efficacy of selective gene silencing of TRIB3 (A) TRIB3 mRNA expression two days after selective gene silencing. Data are presented by using ΔΔCT method regarding peptidyl-prolyl isomerase A (PPIA) normalized TRIB3 mRNA expression of the scrambled (SCR) control as 1 (solid line). Data are expressed as mean±SD of three independent determinations. (B) Western blot analysis of lysates of untransfected (UC), non-sense RNA transfected (SCR: scrambled control), and TRIB3-silenced SZ95 sebocytes two days after the transfection. OD: β-actin-normalized optical density of the corresponding bands. (C) Neutral lipid synthesis (Nile Red staining) following various treatments. Data are expressed as mean±SEM of four independent determinations as the percentage of the SCR vehicle control (100%, solid line). One additional experiment yielded similar results. “siTRIB3a” and “siTRIB3b” mark two different constructs against TRIB3. AA: arachidonic acid, CBD: cannabidiol, TRIB3: tribbles homolog 3.

A B C 2*10-3

1.5*10-3 A2a

= = 1) NC

PPIA expression

of 1*10-3 mRNA

0.5*10-3

expression

( Relative

A2a receptor A2a receptor (ADORA2A) mRNA expression

Supplemental Figure 16. Human SZ95 sebocytes express adenosine A2a receptor (A) Quantitative “real time” PCR on SZ95 sebocyte samples harvested at 60% confluence. Data of A2a receptor (ADORA2A) mRNA expression was normalized to the level of PPIA of the same sample, and is expressed as mean±SD of three independent determinations. Two additional experiments yielded similar results. (B) Immunocytochemistry. A2a-specific immunreactivity was determined by immunofluorescence labeling (Alexa-Fluor®-488, green fluorescence) in SZ95 sebocytes. Nuclei were counterstained with DAPI (blue fluorescence). NC: negative control. Scale bar: 10 μm. (C) Western blot analysis of lysate of SZ95 sebocytes (~60% confluence culture).

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