Figure 5

Ero1α mediates calcium flux from ER to the mitochondria through engagement of MAM. (a) HeLa cells stably expressing ER-targeted ratiometric FRET probe (D1ER chameleon) was used to monitor release of ER calcium. CFP/YFP ratio was measured from images and represented graphically (n=3). The ratio images of cells before PAC-1 treatment and 2 h after PAC-1 treatment are shown along with ratio scale. (b) HeLa D1ER cells were transfected with either scrambled siRNA or siRNA against human Ero1α or CHOP as described. The cells were treated with PAC-1 for 24 h and whole-cell extract was used for western blot using Ero1α or CHOP antibody. β-Actin served as loading control. (c) HeLa D1ER cells 24 h after transfections with siEro1α or siCHOP were treated with PAC-1 and imaged for calcium release as described. The average CFP/YFP ratio was measured from images and represented graphically (n=3). (d) Cells transfected with siSC (scrambled) or siEro1α or siCHOP were treated with PAC-1 for 24 h. Cell death was quantified after staining the cells using Hoechst to visualize chromatin condensation. (e) HeLa, U2OS, SW480, p53 knockout HCT116 cells and MEF Bax/Bak DKO cells were treated with PAC-1 for indicated time periods. The whole-cell extract was prepared and used for western blotting using antibody against Ero1α. Induction of Bim exhibited by MEF Bax/Bak DKO cells is also shown. β-Actin served as loading control. (f) HeLa cells expressing EGFP targeted at ER and DsRed targeted at mitochondria were treated with PAC-1 for 12 h. Representative confocal images of mitochondria and ER of treated and untreated cells are shown