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Supporting Information Khaliullina et al. 10.1073/pnas.1416463112 SI Materials and Methods 10 min. The supernatant containing hemolymph was centri- Mammalian Cell Culture. HeLa cells were passaged in DMEM with fuged at 16,000 × g for 2 h to remove the debris and subjected to 10% FBS (Gibco), 50 U/mL penicillin, and 50 μg/mL strepto- lipid extraction. mycin (Gibco). ShhLIGHT2 cells, which are NIH 3T3 cells that express firefly Lipid Extraction. Lipids from purified VLDL, LDL, HDL, and Lpp luciferase under the control of a Gli-responsive promoter, and particles applied as crude extracts in signaling assays were Renilla luciferase controlled by the viral Thymidine kinase promoter extracted by a two-step Bligh and Dyer method (10) and applied (1) were maintained in DMEM + 10% (vol/vol) FBS, 150 μg/mL to ShhLIGHT2 cells and wing discs at concentrations found in zeocin (Invitrogen), and 400 μg/mL G418 (Invitrogen). hemolymph/human serum. Total lipid concentration was mea- HEK293S-TetR cells, which stably express an inducible SMO sured according to ref. 11. construct, were described (2). Cells were maintained in medium containing DMEM/F-12, 10% (vol/vol) FCS, 2 mg/mL Ge- Preparation of Saponification-Resistant Lipids. Dried VLDL lipid neticin, and 5 μg/mL Blasticidin, as described. extracts were incubated at 80 °C with 2 mL of 0.3 M methanolic NIH 3T3/Smo-mEos2 cells, which are NIH 3T3 cells that stably potassium hydroxide for 1 h. After cooling down to room tem- express Smo-mEos2 construct, were described (3). Cells were perature, the solution was extracted by two washes with diethyl maintained in DMEM + 10% (vol/vol) FCS. ether. The ether fractions, which contain saponification-resistant lipids, were combined and subjected to further analysis. Fly Stocks. Oregon R flies, hs-flippase, and adh-GAL4 obtained from Bloomington Stock Center, transgenic line UAS < HcRed > Preparation of Shh Applied in the ShhLIGHT2 Cell Assay. To prepare dsLpp is described in ref. 4. nonlipoprotein-associated Shh, we transfected HeLa cells with cDNA encoding human Shh (OriGene; SC300021) by using Lipid Extraction from Drosophila Larval Hemolymph. Third instar polyethylenimine (Polysciences) in OptiMEM (Invitrogen), then larvae were washed in 10% (vol/vol) NaCl and disrupted by using switched Shh-transfected HeLa cells to serum-free media + a loose dounce homogenizer, which releases the hemolymph but (DMEM 1% Insulin-Transferrin-Selenium mixture (ITS-X, is not designed to disrupt the cells, in 1% acetic acid in H O with Gibco) 4 h after transfection. After 48 h, conditioned media were 2 × 50 μM PF-3845. The extract was centrifuged at 1,000 × g for centrifuged at 1,000 g for 20 min and concentrated by using 10 min. The supernatant containing hemolymph was centrifuged Amicon Ultra -10K (Millipore) for use in the signaling assay. at 16,000 × g for 2 h to remove the debris and subjected to lipid Identically treated media from nontransfected HeLa cells extraction. were used as controls. To prepare Lipoprotein-Shh, transfected HeLa cells were cultured in DMEM + 10% (vol/vol) FBS. Con- Reagents. Lipids used in this study were as follows: cholesterol, ditioned media were centrifuged at 1,000 × g for 20 min. For desmosterol, 7-dehydrocholesterol, vitamin D3 (Avanti Polar signaling ShhLIGHT2 assay, lipoprotein-Shh was isolated by Lipids), ergosterol, ergocalciferol D2, 1,25-dihydroxyvitamin D3 density centrifugation (12) and concentrated by using Amicon (Sigma-Aldrich), N-acylethanolamides 16:0, 16:1, 18:0, 18:1, Ultra -10K. 18:2, 20:4, N-acyldopamine 16:0, N-acyldopamine 20:4, 2-alkyl- ShhLIGHT2 Signaling and SAG Competition Assays. Twenty-four glycerol 18:2, 2-acylglycerol 20:4, 2-alkylglycerol 20:4 and 5 N-acylserine 20:4 (Cayman Chemical), N-acyl dopamine 18:0 hours before assay, ShhLIGHT2 cells were plated at 10 per well (Tocris Bioscience), N-acyl serine 16:0 (Avanti Polar Lipids), in 96-well plates and then switched to a serum-free medium + delta-9-tetrahydrocannabinol (Biomol), Cannabinol and Cannabidiol consisting of DMEM 1% ITS-X. In this medium, cells were (Sigma-Aldrich), ACEA, AM251, AM404, AM630, CB-13, O-1918, then treated with nontransfected serum-free HeLa cell super- CP-775146, GW6471, PF-3845, S 26948 and T 0070907 (Tocris natants and DMSO or DOPC liposomes (mock) or with non- Bioscience), SMO Agonist (SAG) (Santa Cruz Biotechnology), and lipoprotein-associated Shh supplemented with DMSO or DOPC BODIPY-cyclopamine (BioVision). Deuterated endocannabinoids liposomes (Shh) or with different lipids or synthetic compounds. used for LC-MS/MS quantification of endogenous species were Purified, commercially obtained lipids, or synthetic compounds purchased from Biomol and R&D Systems. were added in DMSO at indicated concentration. HPLC elution fractions were added in DOPC liposomes in Induction of RNAi. LppRNAi was induced as described (4). a ratio of 1:4. SAG was added to the ShhLIGHT2 cells at indicated concen- Immunohistochemistry and Image Analysis. Imaginal discs were trations alone or in the presence of different endocannabinoids. fixed and stained as described (5). Image analysis and statistics of Luciferase activity was assayed in cell lysates after 24 h, as image quantifications were performed as described (6). Anti- instructed by the manufacturer (Dual Glo Luciferase Assay; bodies were diluted as follows: anti-Ci 2A1 1:10 (7) and anti- Promega). The resulting Hh pathway activity was measured as the SMO 1:50 (8). firefly:Renilla ratio normalized to the ratio in cells that were not stimulated with Shh. Lipoprotein Isolation. VLDL, LDL, and HDL were isolated from human serum (Sigma) according to ref. 9. Drosophila Lipophorin BODIPY-Cyclopamine Binding Assay. HEK293S-TetR cells were (Lpp) was isolated as described (6). grown to confluence in medium containing Geneticin (2 mg/mL). The growth medium was then replaced with fresh medium Lipid Extraction from Drosophila Larval Hemolymph. Third instar containing tetracycline (2 μg/mL) and sodium butyrate (5 mM). larvae were washed in 10% (vol/vol) NaCl and disrupted by using After 2 d, fluorescence binding assays using BODIPY-cyclop- a loose dounce homogenizer, which releases the hemolymph but amine were conducted as described (13). BODIPY fluores- is not designed to disrupt the cells, in 1% acetic acid in H2O with cence was monitored by FACS and analyzed by using the 50 μM PF-3845. The extract was centrifuged at 1,000 × g for FlowJo software. Khaliullina et al. www.pnas.org/cgi/content/short/1416463112 1of9 Ciliary SMO Localization Assay. NIH 3T3/Smo-mEos2 cells were normal goat serum), incubated with the anti-acetylated Tubulin grown on glass coverslips to reach confluence. The cells were then antibody (Sigma, 1:2,000) in blocking solution for 1 h, washed with shifted to DMEM + 1% ITS-X and incubated for 24 h with or PBS, incubated with the secondary antibody in blocking solution, without SAG and different compounds. Cells were fixed for washed, and mounted in Vectashield. Slides were viewed under 10 min with 4% (vol/vol) formaldehyde in PBS, incubated for the confocal microscope Zeiss LSM Meta and analyzed by using 20 min in blocking solution (PBS + 0.1% Triton X-100 + 1% the Fiji software. 1. Taipale J, Cooper MK, Maiti T, Beachy PA (2002) Patched acts catalytically to suppress 8. Lum L, et al. (2003) Hedgehog signal transduction via Smoothened association with the activity of Smoothened. Nature 418(6900):892–897. a cytoplasmic complex scaffolded by the atypical kinesin, Costal-2. Mol Cell 12(5): 2. Dwyer JR, et al. (2007) Oxysterols are novel activators of the hedgehog signaling 1261–1274. pathway in pluripotent mesenchymal cells. J Biol Chem 282(12):8959–8968. 9. Redgrave TG, Roberts DC, West CE (1975) Separation of plasma lipoproteins by 3. Kim J, et al. (2014) Simultaneous measurement of smoothened entry into and exit density-gradient ultracentrifugation. Anal Biochem 65(1-2):42–49. from the primary cilium. PLoS ONE 9(8):e104070. 10. Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. 4. Panáková D, Sprong H, Marois E, Thiele C, Eaton S (2005) Lipoprotein particles are Can J Biochem Physiol 37(8):911–917. required for Hedgehog and Wingless signalling. Nature 435(7038):58–65. 11. Marsh JB, Weinstein DB (1966) Simple charring method for determination of lipids. J 5. Marois E, Mahmoud A, Eaton S (2006) The endocytic pathway and formation of the Lipid Res 7(4):574–576. Wingless morphogen gradient. Development 133(2):307–317. 12. Rudel LL, Lee JA, Morris MD, Felts JM (1974) Characterization of plasma lipoproteins 6. Khaliullina H, et al. (2009) Patched regulates Smoothened trafficking using lipopro- separated and purified by agarose-column chromatography. Biochem J 139(1):89–95. tein-derived lipids. Development 136(24):4111–4121. 13. Chen JK, Taipale J, Cooper MK, Beachy PA (2002) Inhibition of Hedgehog signaling by 7. Wang QT, Holmgren RA (1999) The subcellular localization and activity of Drosophila direct binding of cyclopamine to Smoothened. Genes Dev 16(21):2743–2748. cubitus interruptus are regulated at multiple levels. Development 126(22):5097–5106. Khaliullina et al. www.pnas.org/cgi/content/short/1416463112 2of9 Fig. S1. Lipids derived from mammalian lipoproteins reduce SMO levels in Drosophila wing imaginal discs and inhibit Shh signaling in ShhLIGHT2 cells. (A) Ratio of firefly:Renilla luciferase activity in ShhLIGHT2 cells treated as indicated. Treatment with DMSO served as a control for background activity (-). Error bars indicate SDs of three independent experiments. n > 3. (B′–B****) Wing discs from wild-type (B′) and LppRNAi (B*–B****) animals mock-treated (B′ and B*) or treated with HDL- (B**), LDL- (B***), or VLDL-derived (B****) lipids. (C) Quantification of SMO staining in the wing disk assay performed in (B′–B****) with lipids derived from HDL (magenta; P = 1.4), LDL (blue; P = 0.0048 for far anterior) or VLDL (green; P = 1.0 × 10−11).
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