SUPPLEMENTAL INFROMATION

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1 SUPPLEMENTAL INFROMATION SUPPLEMENTAL EXPERIMENTAL PROCEDURES IP 1 accumulation - IP 1 accumulation was quantified using the HTRF IP-One kit (Cisbio Bioassys, Bedford, MA, USA) according to the manufacturer s instructions. Briefly, T6.11 cells were seeded at 25 cells/well in a 384-well plate and then treated with PI3K inhibitors for 2 min at 37 C before being stimulated with 5 µm CCh for 3 min. A lysis medium containing an anti-ip 1 antibody labeled with Lumi4-Tb and an IP 1 -d2 derivative was added to the cells. After 1 h at room temperature, the time-resolved FRET signals were measured after excitation at 665 and 62 nm using a Tecan M1 instrument. The data represent IP 1 levels in pmol/25 cells calculated from the calibration curve generated according to the manufacturer s instructions. Lipids extraction and quantification of PI(4,5)P 2 and PI(3,4,5)P 3 Lipids were extracted as described by Grey et al (23) 1. Briefly, cells were incubated with cold.5 M TCA for 5 min, centrifuged and resuspended in 5% TCA/1 mm EDTA. After centrifugation, neutral lipids were extracted with methanol:chloroform (2:1) and acidic lipids with methanol:chloroform:12 M HCl (8:4:1). The extracts were centrifuged and chloroform plus.1 M HCl were added to the supernatant followed by centrifugation to separate organic and aqueous phases. The organic phase was dried in a vacuum dryer. For PI(4,5)P 2 quantification, T6.11 cells plated in 6-well plates at 3x1 5 cells/well were grown for 24 h. PI(4,5)P 2 was quantified using the PI(4,5)P 2 Mass ELISA kit (Echelon Biosciences, Salt Lake City, UT, USA) following the manufacturer s instructions. Briefly, extracted lipids were resuspended in PBS.25% Protein Stabilizer, incubated with a PI(4,5)P 2 detector protein for 1 h at room temperature and then added to a 96- well PI(4,5)P 2 -coated microplate. A peroxidase-linked secondary detection reagent and a colorimetric substrate were added to detect the PI(4,5)P 2 detector protein binding to the plate. The colorimetric signals were measured at 45 nm using a Tecan M1 instrument. The data represent PI(4,5)P 2 levels in pmol/1 cells calculated from the standard curve generated according to the manufacturer s instructions. For PI(3,4,5)P 3 quantification, PIP 3 was extracted from 5 x 1 6 T6.11 cells. Extracted lipids were resuspended in 5% DMSO, sonicated and spotted onto nitrocellulose membranes. The dot membranes were blocked in TBST (2 mm Tris-HCl, ph 7.5, 137 mm NaCl,.1% Tween 2) with 5% nonfat dried milk for 1 hour at room temperature and then were probed with a mouse anti-pip 3 antibody (1:5) (MBL International), followed by peroxidase-conjugated sheep anti-mouse-igg (1:1 ). Immune complexes were detected using Western Lightning Chemiluminescence Reagent Plus kits according to the manufacturer s instructions and quantified by densitometric analysis. 1. Grey, A. Olsson, H., Batty, I.H., Priganica, L., Downes, C.P. (23) Analytical Biochemistry 313, Supplemental information, Monet et al. 1

2 SUPPLEMENTAL FIGURE LEGENDS Supplemental Figure S1. Effect of wortmannin and PIK-93 on cellular level of PIP 2. Using a competitive ELISA (see supplemental Experimental Procedures), PI(4,5)P 2 was quantified in cells pretreated with wortmannin (1 nm and 1 µm) and PIK-93 (3 nm) for 2 min at 37 C. Cells were stimulated or not with 5 µm CCh for 5 min at 37 C. PI(4,5)P 2 levels are expressed in pmol/1 cells and represent the means ± SD of three independent experiments. P <.5. Supplemental Figure S2. Effect of wortmannin and PIK-93 on CCh-induced inositol phosphate accumulation. IP 1 production was measured using an immunocompetitive HTRFbased assay (see supplemental Experimental Procedures). T6.11 cells were pretreated for 2 min at 37 C with wortmannin (1 nm and 1 µm) or PIK-93 (3 nm) before being stimulated or not with 5 µm CCh for 3 min at 37 C. IP 1 accumulation is expressed in pmol/25 cells and represents the means ± SD of three independent experiments. P <.5. Supplemental Figure S3. Knockdown of PTEN enhance the basal level of membrane PIP 3. Measurement of PIP 3 was evaluated by dot blot assay (see supplemental Experimental Procedures). Densitometric analysis of the immunoreactive areas was performed and the results are expressed as fold PIP 3 level in sictl-treated cells. Bars represent means ± SD of three independent experiments. P <.5. Supplemental information, Monet et al. 2

3 PI(4,5)P 2 (pmol/1 cells) CCh (5 µm) Supplemental figure S1, Monet et al.

4 IP 1 (pmol/25 ce ells) CCh (25 µm) 3,5 CCh (5 µm) 3, 2,5 2, 1,5 1,,5 Supplemental figure S2, Monet et al.

5 Standard PIP 3 (fmol) PIP P 3 (fold Ctl level) sictl sipten Supplemental figure S3, Monet et al.