6. Clone, . B4, and . Bioscience, USA) was performed by addition of saponin, FBS and BSA to a final concentration of 0.1%, 2%, and 1% (w/v), respectively, followed by storage overnight at 4 1C. The median fluorescence intensity (MFI) of the cyto-c staining was normalized to isotype control and inert peptide (PUMA2A) controls. The percentage of cyto-c release was calculated as following: % cyto-c release ¼ 100

, xL cells were infected with H2B-RFP lentivectors (MOI ¼ 3applicable, shCt or shBcl-xL lentivectors were added for 48 h before the double block thymidine was made. Finally, cells were released 1 h before addition of drugs and analysis. Imaging was performed using a Leica DMI 4000 Bioimager (Leica, Images were collected every 10 min using a  10 objective. Image sequences were viewed using NIH ImageJ software, and cell behavior was analysed manually

. Immunoblotting, 10 mg/ml leupeptin, 10 mg/ml pepstatin, 1 mM Na3VO4, 50 mM NaF and sonication. SDS-PAGE were run on 12.5% polyacrylamide gel and proteins transferred to a PVDF membrane. The following antibodies were used at the indicated dilution: Bcl-2 (MO887, 1 : 500, Protein extracts were obtained after cell lysis at 4 1C in buffer containing 50 mM Tris-HCl, pH 8.1, 1% SDS, 10 mM EDTA, 1 mM PMSF, 10 mg/ml aprotinin, 1000.

. Rt-qpcr, ) at a final concentration of 5 mM, as described by Vo et al. 34 The BRET signal (mB.U, milliBRET Unit) was determined by calculating the ratio of the light emitted by the acceptor fusion protein eYFP-Bcl-xL (530 nm) over the light emitted by the donor fusion protein Rluc-Bax (475 nm). The values are corrected by subtracting the background signal detected when the donor constructs is expressed with the nontagged acceptor Bcl-xL. For dose-response effect of ABT-737, %BRET is calculated using the following formula: (BRET meas -BRET min ) Â 100/(BRET max -BRET min ), where BRET meas is the BRET ratio obtained for every ABT737 concentration, Then 500 ng of total RNA was reverse transcribed by using the superscript III reverse transcriptase and random hexamers (Life Technologies). Quantitative PCR was done by using the Maxima SYBR Green/ROX qPCR Master Mix (Life Technologies) and the MX4000 instrument

, MCF-7 cells were plated onto glass coverslips and grown for 24 h before transfection with peYFP-Bcl-xL, pS62A-eYFP-Bcl-xL or pS62D-eYFP-Bcl-xL plasmids. Forty-eight hours after transfection, cells were stained with the cell-permeant MitoTracker probe (MitoTracker Red CMXRos, Invitrogen) as described by the manufacturer, Slides were then counterstained with DAPI and images were viewed on a Zeiss Axiovert 200 M microscope (MicroPIcell imaging facility)

, Yeast-expressing Bax and S62A or S62D-Bcl-xL were obtained by transforming the wild-type haploid strain BY4742 (mata his3 leu2 lys2 ura3) using pRS415-GPD-Bax plasmid under the control of the inducible GAL1/10 promoter (LEU2 as yeast selection marker) and p413-GPD-S62A-Bcl-xL or p413-GPD-S62D-Bcl-xL, coding for S62A-Bcl-xL or S62D-Bcl-xL, respectively

. Alternatively, Cells were grown aerobically at 30 1C in a synthetic medium containing 0.67% yeast nitrogen base with ammonium sulfate (Difco), 0.01% of auxotrophic requirements, and 2% glucose as a carbon source on Petri dishes for 48 h or alternatively 2% raffinose and 2% galactose in 96-well plates with a starting cell density of 0.01 OD 595 U/ml, in the presence of ABT-737 at 20 mM, or not. Cell density was measured at 24 h by OD 595 and ratios between ABT-737-treated and untreated conditions were calculated for each Bcl-xL mutant, Bax was expressed from the constitutive GPD promoter into the p415-GPD plasmid (LEU2 as yeast selection marker), p.28

, Statistical analysis was performed by using one-tailed Student's t-test on R. Errors bars represent standard errors of the mean, Statistics

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