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4 protocols using 1 oleoyl 2 acetyl sn glycerol

1

Synthesis and Characterization of Calcium Signaling Modulators

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([(3S)‐6‐({2′,6′‐dimethyl‐4′‐[3‐(methylsulfonyl)propoxy]biphenyl‐3‐yl}methoxy)‐2,3‐dihydro‐1‐benzofuran‐3‐yl]acetic acid hemihydrate) was synthesized at Takeda Pharmaceutical Company (Osaka, Japan). Diazoxide, KCl, and glimepiride were purchased from Wako Pure Chemical Industries (Osaka, Japan). Sterile D‐(+)‐glucose solution (45% in water), γ‐linolenic acid (γ‐LA), and (S)‐(‐)‐Bay K8644 were purchased from Sigma‐Aldrich (St. Louis, MO). D‐myo‐inositol 1,3,5‐trisphosphate hexakisacetoxymethyl ester, 2,4,6‐tri‐O‐butyryl (Bt3IP3), a cell‐permeable derivative of D‐myo‐inositol 1,3,5‐trisphosphate, was purchased from Merck Millipore (Tokyo, Japan). A cell‐permeable analog of DAG, 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG), was purchased from Cayman Chemical (Ann Arbor, MI). Nifedipine was purchased from MP Biomedicals (Tokyo, Japan). Krebs‐Ringer‐bicarbonate‐HEPES (KRBH) buffer (116 mmol/L NaCl, 4.7 mmol/L KCl, 1.17 mmol/L KH2PO4, 1.17 mmol/L MgSO4, 25 mmol/L NaHCO3, 2.52 mmol/L CaCl2, and 24 mmol/L HEPES (pH 7.4)) containing 0.2% bovine serum albumin (BSA) was freshly prepared before each experiment. Fura‐2 acetoxymethyl ester was purchased from Dojindo Laboratories (Kumamoto, Japan).
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2

Lipid Compound Characterization and Storage

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The following chemicals were obtained from Cayman Chemical: linoleic acid (#90150), 2-linoleoyl glycerol (#62260), linoleoyl ethanolamide (#90155), linoleoyl glycine (#9000326), oleic acid (#9000326), 1-oleoyl-2-acetyl-sn-glycerol (OAG, #62600), 2-arachidonoyl glycerol (#62160), 2-arachidonoyl glycerol ether (#62165), R-1 methanandamide (#90070), JZL 184 (#13158) and IDFP (#10215). The chemicals were dissolved in ethanol or DMSO and kept at −80°C. Riluzole [2-Amino-6-(trifluoromethoxy)benzothiazole] (329-92191, FUJIFILM Wako Chemicals) and GSK1702934A (SML2323, Sigma-Aldrich) were dissolved in DMSO and kept at −20°C. Ruthenium red (R2751, Sigma-Aldrich) was dissolved in water and stored at −20°C.
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3

Pharmacological Inhibition of TRP Channels

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The pan-TRP channel inhibitor 1-[2-(4-methoxyphenyl)]-2-[3-(4-meth-oxyphenyl)propoxy]ethyl-1H-imidazole hydrochloride (SKF-96365), the ROS scavenger 1-oxyl-2,2,6,6-tetramethyl-4-hydroxypiperidine (TEMPOL), and guanosine 5′-[β-thio]diphosphate (GDP-βS) were obtained from Sigma. 20-HETE and 1-oleoyl-2-acetyl-sn-glycerol (OAG) were purchased from Cayman Chemical Co.
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4

Steroid Production in WT MA-10 and STAR KO Cells

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WT MA-10 and STAR KO cells (1×104 per well) were plated on 96-well plates in triplicate for 24 h. Before stimulation, medium was removed, each well was washed with phosphate-buffered saline, and serum-free medium was added with one of the following: 50 ng/mL human chorionic gonadotropin (hCG; National Hormone and Peptide Program, Harbor-UCLA Medical Center, Torrance, CA, USA), 1 mM dibutyryl cyclic AMP (dbcAMP; Sigma), 50 μM 22(R)-hydroxycholesterol (Sigma, St. Louis, MO, USA), 50 μM XBD173 (Sigma), 50 μM FGIN-1-27 (Cayman Chemical, Ann Arbor, MI, USA), 1-oleoyl-2-acetyl-sn-glycerol (Cayman Chemical, Ann Arbor, MI, USA), U73122 (Cayman Chemical, Ann Arbor, MI, USA), or 1 mM calphostin C (Cayman Chemical, Ann Arbor, MI, USA). After 2 h incubation at 37 °C, the medium was collected to measure steroid production. The remaining cells were lysed with 0.1 N sodium hydroxide for protein measurements. Steroid production was measured using the Progesterone ELISA Kit (Cayman Chemical, Ann Arbor, MI, USA). Steroid production data were normalized to protein contents.
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