Figure EV2. Existing agents do not protect from ceramide‐induced mitochondrial fission.

• A
  After a 1‐h pre‐treatment with vehicle (DMSO) or mdivi‐1 and M1 (50 μM, 5 μM) for 1 h or 24 h, MEFs were treated for 3 h with vehicle (ethanol) or C16‐CER (100 μM) and stained for citrate synthase.
• B–D
  Aspect ratio (B), branch length (C), or roundness (D) of mitochondria in the cells in (A).
• E–H
  As in (A‐D), but in MEFs pre‐treated with vehicle (methanol) or leflunomide (50 μM) for 1 h or 24 h.
• I–L
  As in (A‐D), but in MEFs pre‐treated with vehicle (water) or P110 (10 μM) for 1 h or P110 (1 μM) for 12 h.
• M
  MEFs treated for 3 h with BSA or palmitate (250 μM) after a 3‐h pre‐treatment with vehicle (DMSO), celastrol (500 nM), or withaferin A (WFA, 500 nM).
• N–P
  Aspect ratio (N), branch length (O), or roundness (P) of mitochondria in the cells in (M).
• Q
  MEFs were pre‐treated with vehicle (water) or SH‐BC‐893 for 3 h and then treated with vehicle (DMSO) or FCCP (1 μM) for 1 h.
• R–T
  Aspect ratio (R), branch length (S), or roundness (T) of mitochondria in the cells in (Q).

Data information: In B‐D, F‐H, J‐L, N‐P, and R‐T, 40 cells from 2 biological replicates were evaluated and mean ± SD shown. Using a one‐way ANOVA with Tukey’s correction (D, G, H, P, and T) or Brown–Forsythe and Welch ANOVA tests with Dunnett’s correction for multiple comparisons (B, C, F, J‐L, N, O, R, and S), ***P ≤ 0.001; **P ≤ 0.01; *P ≤ 0.05; ns, not significant, P > 0.05. Scale bars, 20 µm.