Supporting Information Ohno et al. 10.1073/pnas.1503491112 SI Materials and Methods hexane. Methanol/water and hexane phases were separated by Cell Culture and Transfection. HeLa and HEK 293T cells were cul- centrifugation, and the hexane (upper) phase was recovered. tured in DMEM (Sigma) supplemented with 10% FBS, 100 U/mL After hexane extraction was repeated three times, the extracted penicillin, and 100 μg/mL streptomycin. HEK 293T cells were lipids were combined, dried, and suspended in chloroform/ grown in dishes precoated with 0.3% collagen. Transfections methanol (2:1, vol/vol). Lipids were separated by reverse-phase were performed using Lipofectamine Reagent and Plus Re- TLC with Silica Gel 60 RP-18 F254s TLC plates (Merck Milli- agent (Life Technologies), according to the manufacturer’s pore) with chloroform/methanol/water (15:15:2, vol/vol/vol) and protocols. Normal human epidermal keratinocytes isolated detected as described above. from juvenile donors were purchased from CELLnTEC and grown in PCT Epidermal Keratinocyte Medium (CELLnTEC). Lipid Analysis by MS. To examine ceramide and FA species pre- Keratinocyte differentiation was induced by exchanging the pared from cultured cells, lipids were analyzed by UPLC coupled medium to Epidermal Keratinocyte 3D Prime Medium with ESI tandem triple quadrupole MS (Xevo TQ-S; Waters). (CELLnTEC) after the cell confluency reached ≥80%. Cells were washed twice with 1 mL of PBS, suspended in 1 mL of The Saccharomyces cerevisiae strain BY4741 (MATa his3Δ1 PBS, and detached from culture dishes by pipetting. Cell sus- leu2Δ0 met15Δ0 ura3Δ0) (36) was used. Cells were grown in pensions were transferred to silicon-coated plastic tubes. After × synthetic complete (SC) medium (0.67% yeast nitrogen base and centrifugation (400 g at room temperature for 3 min), cells μ 2% D-glucose) containing 0.5% casamino acids, 20 mg/mL ade- were suspended in 100 L of PBS, followed by the successive μ nine, and 20 mg/mL tryptophan but lacking uracil (SC-URA). addition and mixing of 375 L of chloroform/methanol/12 M formic acid (100:200:1, vol/vol/vol), 2 μLof25μM C17:0 ce- Plasmids. The pCE-puro 3xFLAG-ELOVL4 plasmid encoding ramide (internal standard; Avanti Polar Lipids), 125 μLofchlo- N-terminally 3xFLAG-tagged ELOVL4 has been described pre- roform, and 125 μL of water. After centrifugation (9,000 × g at viously (29). Human CYP4F subfamily genes and the human room temperature for 1 min), the organic phase was recovered CERS3 gene were amplified by PCR using their respective for- and dried. Lipids were suspended in 200 μL of chloroform/ ward and reverse primers listed in Table S2. The amplified DNAs methanol (1:2, vol/vol) and were mixed with 6 μLof4MKOH. first were cloned into pGEM-T Easy Vector (Promega) and then After incubation for 1 h at 37 °C, lipids were mixed with 7 μLof were transferred to the pCE-puro 3xFLAG-1 plasmid, a mam- 4 M formic acid, 66.6 μL of chloroform, and 133.3 μL of water. malian expression vector designed to produce an N-terminal After centrifugation (9,000 × g at room temperature for 1 min), 3xFLAG-tagged protein. The CYP4F22 gene was also transferred the organic phase was recovered, dried, and dissolved in 50 μLof to the pAK1017 plasmid (URA3 marker, CEN), a yeast expression chloroform/methanol (1:1, vol/vol). As controls, epidermal lipids vector designed to produce an N-terminal, tandemly oriented were prepared from mice as described previously (29, 38). Lipids His6-, Myc-, and 3xFLAG-tagged protein under the control of the were resolved by UPLC on a reverse-phase column (ACQUITY TDH3 (glyceraldehyde 3-phosphate dehydrogenase) promoter, UPLC BEH C18 column, length 150 mm; Waters) at 45 °C and creating the pNS29 plasmid. Ichthyosis mutations were introduced were detected by MS. The flow rate was 0.1 mL/min in the binary into the CYP4F22 gene using the QuikChange Site-Directed gradient system using a mobile phase A [acetonitrile/water/metha- Mutagenesis Kit (Agilent Technologies), using the primers de- nol (4:4:2, vol/vol/vol) containing 0.1% formic acid and 0.025% scribed in Table S2. The N-glycosylation site cassette was inserted ammonia] and a mobile phase B [2-propanol/methanol (4:1, vol/vol) into the CYP4F22 gene as described previously (37), using the containing 0.1% formic acid and 0.025% ammonia]. The elution T (topology)-series primers listed in Table S2. gradient steps were as follows: 0 min, 5% B; 0–10 min, gradient to 60% B; 10–50 min, gradient to 80% B; 50–55 min, gradient to 100% [3H]Sphingosine Labeling Assay. HEK 293T cells were transfected B; 55–65 min, 100% B; 65–66 min, gradient to 5% B; 66–80 min, with appropriate plasmids. Twenty-four hours after transfection, 5% B. The ESI capillary voltage was set at 3.0 kV; the sampling cells were labeled for 4 h at 37 °C with 2 μCi [3-3H]sphingosine cone was set at 30 V; and the source offset was set at 50 V in (20 Ci/mmol; PerkinElmer Life Sciences). Cells were washed positive ion mode. Each ceramide species was detected by MRM by + + twice with 1 mL of PBS and suspended in 100 μL of PBS. Lipids selecting the m/z ([M−H2O+H] and [M+H] ) of specific ceramide were extracted by successive addition and mixing of 375 μLof species at Q1 and the m/z 264.2 at Q3 (Table S3). Data analysis and chloroform/methanol/HCl (100:200:1, vol/vol/vol), 125 μL of chlo- quantification were performed using MassLynx software (Waters). roform, and 125 μL of 1% KCl. Phases were separated by centri- For quantitation of FAs in keratinocytes, lipids were extracted fugation (20,000 × g at room temperature for 3 min), after which and treated with an alkali as described above. As an internal stan- the organic (lower) phase was recovered, dried, and subjected to dard, C13:0 FA (0.2 pmol; Sigma) was added. Extracted lipids were + normal-phase TLC (Silica Gel 60 TLC plates; Merck Millipore) dried, derivatized by an AMP Mass Spectrometry Kit (Cayman with the following solvent systems: (i) chloroform/methanol/water Chemical) according to the manufacturer’s protocol, and resolved (40:10:1, vol/vol/vol), developed to 2 cm from the bottom of the by UPLC-ESI MS essentially as described above except as outlined TLC plate, dried, and then developed again to 5 cm from below. The flow rate was set at 0.15 mL/min and the elution gra- the bottom; (ii) chloroform/methanol/acetic acid (47:2:0.5, vol/vol/vol), dient steps were set as follows: 0 min, 5% B; 0–3min,gradientto developed to the top; and (iii) hexane/diethylether/acetic acid 60% B; 3–10 min, gradient to 80% B; 10–11 min, gradient to 100% B; (65:35:1, vol/vol/vol), developed to the top twice. Labeled lipids 11–20 min, 100% B; 20–21 min, gradient to 5% B; 21–30 min, were visualized by spraying the plate with a fluorographic re- 5% B. Hydroxy FA species were detected by MRM by selecting the + agent [2.8 mg/mL 2,5-diphenyl-oxazole in 2-methylnaphthalene/ m/z ([M+H] ) of the derivatized hydroxy FA species at Q1 and the 1-buthanol (1:3.3, vol/vol)]. The TLC plate was exposed to X-ray m/z 238.9 at Q3, corresponding to the fragment cleaved between C3 film at −80 °C. and C4 of derivatized FAs (Table S3). For reverse-phase TLC analysis, extracted lipids were sus- To examine ceramide species in the stratum corneum of human pended in 150 μL of 90% methanol and mixed with 150 μLof subjects, tape stripping was performed by pressing an acryl film tape Ohno et al. www.pnas.org/cgi/content/short/1503491112 1of11 (465#40; Teraoka Seisakusho) to the skin of the forearm. Five (39), using anti-FLAG M2 (10 μg/mL; Sigma), anti-calnexin 4F10 strips, measuring 25 mm × 50 mm each, were obtained from a (10 μg/mL; Medical & Biological Laboratories), or anti-HA HA-7 single person. The tapes were immersed in 3.0 mL methanol with (50 μg/mL; Sigma) antibody as a primary antibody and Alexa μ 60 L 500 nM C17:0 ceramide as an internal standard. After 10 min Fluor 488-conjugated anti-rabbit antibody or Alexa Fluor 594- of sonication, the lipid extracts were dried under a nitrogen stream conjugated anti-mouse antibody (each at 5 μg/mL; Molecular and then were dissolved in chloroform/methanol/2-propanol Probes, Life Technologies) as a secondary antibody. Coverslips (10:45:45, vol/vol/vol) so that the final concentration of the in- ternal standard was 50 nM. This lipid solution was subjected to were mounted with Prolong Gold Antifade Reagent (Molecular reversed-phase LC/MS. An Agilent 1100 Series LC/MSD SL Probes, Life Technologies) and observed under a Leica DM5000B system equipped with a multi-ion source, ChemStation software, microscope (Leica Microsystems). a 1,100-well plate autosampler (Agilent Technologies), and an L-column ODS (2.1 mm i.d. × 150 mm; Chemicals Evaluation and Deglycosylation. Protein deglycosylation was performed using Research Institute) was used. Chromatographic separation of endoglycosidase H (New England Biolabs), according to the ’ the lipids was achieved at a flow rate of 0.2 mL/min using a bi- manufacturer s instructions. nary gradient solvent system of mobile phase C [methanol/ water ω (1:1, vol/vol) containing 5 mM acetic acid and 10 mM ammo- In Vitro FA -Hydroxylase Assay. Total membrane fractions were nium acetate] and mobile phase D (2-propanol containing 5 mM prepared from yeast cells as described previously (40) and were acetic acid and 10 mM ammonium acetate).