3h inositol

As a positive control, Saccharomyces cerevisiae (w303 strain) was grown in 2 ml complete synthetic medium with 30 µCi/ml ³[H]-inositol (Perkin Elmer) overnight at 30°C to reach saturation. Half of the cultures were osmotically shocked by adding KCl (1M) for 10 min to induce PI(3,5)P₂ synthesis. Lipids from the harvested yeasts were extracted and deacylated (Bird, 1994 (link)). For MDA-MB-231 cells, 5 × 10⁵ cells/60-mm dish were seeded and grown at 37°C overnight before washing with inositol-free DMEM-H medium (MP Biomedicals), and growing in 5 ml of inositol-free DMEM-H with 30 µCi/ml ³[H]-inositol and 10% dialyzed FBS for 3 d to reach full confluence. Cellular lipids were then extracted and deacylated. The deacylated lipids were analyzed by HPLC on a Partisphere SAX column (4.6 × 125 mm; Whatman) using the following buffer profile: 10 mM NH₄H₂PO₄ (pH 3.5, buffer A) for 7 min, 0–7% buffer B (1.7 M NH₄H₂PO₄, pH 3.5) in the next 3 min, 7–14% buffer B in the next 15 min, then 14–60% buffer B for the next 10 min, and followed by 60–100% buffer B for another 15 min. The flow rate throughout the gradient was 1 ml/min, and 1-ml fractions from the column were quantified by scintillation counting.

C18 fatty acids (stearic acid, oleic acid, linoleic acid and α-linolenic acid), verapamil, 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), bisindolylmaleimide (BIM), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and o-phthaldialdehyde (OPT) were obtained from Sigma Chemical (St. Louis, MO, USA). Fura-2/AM was purchased from Enzo Life Science (Farmingdale, NY, USA). [³H] Inositol was obtained from PerkinElmer (Waltham, MA, USA). AG 1-X8 resin was purchased from BIO-RAD (Hercules, CA, USA). The materials for cell culture were purchased from Life Technologies (Grand Island, NY, USA). Unless otherwise stated, all reagents were of the highest purity and were purchased from Sigma Chemical.