Figure 2.
Mfn2 ablation prevents caspase activation during ER stress and promotes paraptosis-like cell death. (A) WT and Mfn2 KO cells were treated with 1 μM Tg for 12 or 24 h. Total and cleaved caspase 3 levels were detected by western blot. Data are mean±s.e.m. (n=3). *P<0.05 versus WT group. (B, C) WT and Mfn2 KO cells were treated with 0.5 μg/ml tunicamycin (Tm), 100 ng/ml brefeldin A (Bref), or 1 μM Tg for 24 h. Total and cleaved caspase 3 levels were detected by western blot (B) and caspase activity (C) by measurement of DEVD-AFC substrate processing. Data are mean±s.e.m. (n=3). *P<0.05 versus WT group. (D) Flow-cytometry analysis of the Sub G1 DNA fragmentation in methanol-fixed WT and Mfn2 KO cells after incubation with or without 1 μM Tg for 24 h. Data are given as mean±s.e.m. (n=3). *P<0.05 versus WT group. (E) Scr (stably expressing scrambled shRNA) and Mfn2 knockdown (KD) (stably expressing shRNA directed against Mfn2) 3T3-L1 fibroblasts were incubated in the presence or absence of 1 μM Tg for 24 h. Total and cleaved caspase 3 were detected by western blot. Data are mean±s.e.m. (n=3). *P<0.05 versus WT group. (F) WT and Mfn2 KO cells were treated with 1 μM Tg for 24 h. Lactate dehydrogenase (LDH) release was analyzed by flow cytometry to assess necrotic cell death. Data are mean±s.e.m. (n=3). *P<0.05 versus WT group. (G) WT and Mfn2 KO cells were incubated for 24 h with or without 1 μM Tg in the presence or absence of z-VAD-fmk and stained for annexin V/PI. Data are mean±s.e.m. (n=4). *P<0.05 versus WT+Tg group. (H) Mfn2 KO cells transfected with the pEGFP plasmid were incubated with 1 μM Tg alone or in combination with 2 μM CHX for 24 h (3 h of pre-incubation with CHX). Florescence microscopy images show that CHX prevents cytoplasmic vacuolization. Scale bar: 10 μm. (I) WT (black circles) and Mfn2 KO cells (white circles) were incubated with 1 μM Tg for varying times, and ALIX protein was detected by western blot. Data are mean±s.e.m. (n=3). *P<0.05 versus WT group.
Source data for this figure is available on the online supplementary information page.
