Figure 6.

PERK is required to maintain ER-mitochondrial contact sites and for phox-ER stress induced relocation of mitochondria to the ER. (A) Confocal analysis of untreated (Ctr) or phox-treated (1 J/cm²) PERK^+/+ and PERK^−/− MEFs. At the indicated time points, cells were fixed and co-stained for calreticulin (ER marker, red), TOM20 (mitochondrial marker, green) and nucleus (blue). Bars represent 20 μm. (B) Manders' colocalization analysis for data represented in (a–f). Graph represents mean±S.D. (n=3, at least 25–30 cells were analyzed per condition). (C) Confocal analysis of PERK^+/+ and PERK^−/− MEFs transiently transfected with either empty vector (pcDNA) or a kinase-dead (PERK-K618A, left panel) or C-terminal deletion mutant (PERK ΔC, right panel) of PERK. Seventy two hours after transfection, cells were fixed and co-stained for calreticulin (ER marker, red), TOM20 (mitochondrial marker, green) and nucleus (blue). Bars represent 20 μm. (D) Western blot analysis of PERK K618A and PERK ΔC expression levels in PERK^−/− MEFs. Seventy two hours after transfection, whole cell lysates were immunoblotted for total PERK. Asterisk (*) indicates an unspecific band. (E) Manders' colocalization analysis for data represented in (A) (panels a and d) and (C) . Graph represents mean±S.D. (n=3, at least 25–30 cells were analyzed per condition). (f) Dissipation of mitochondrial membrane potential (Δψ_m) following phox-ER stress in PERK^+/+, PERK^−/− and PERK^−/− MEFs 72 h after transfection with either empty vector or a kinase-dead (PERK K618A) or C-terminal deletion (PERK ΔC) mutant of PERK. Cells were left untreated (Ctr) or phox-treated (1.62 J/cm²). After 6 h, cells were incubated with 2 nM DiOC6(3) and the percentage of cells with reduced membrane potential (DiOC6(3)−) was determined by flow cytometry. Note that the increased cell density used in this set of experiments attenuated the magnitude of Δψ_m after phox-ER stress compared with that reported in Figure 5d. Graph represents the mean±S.D. of three independent experiments performed in duplicate. (*) indicates P<0.05, (**) P<0.01 and (***) P<0.001 under Student's t-test