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. Author manuscript; available in PMC: 2021 Jun 22.
Published in final edited form as: Dev Cell. 2020 Jun 5;53(6):627–645.e7. doi: 10.1016/j.devcel.2020.05.014

Figure 4. Interfering with EL Targeting of Mitochondria Through KD of Rab5 Uncouples BAX Clustering at OMMs from Cytochrome c and Smac Release.

Figure 4.

(A) Immunoblot of Rab5 levels in MCF-7 cells stably expressing shCON, shRab5A or shRab5C. GAPDH, loading control.

(B) Cells as in (A), treated with STS for 4 hrs, IF of BAX and TOM70.

(C) Quantifications of cells as in (B) in FM, or treated with STS for 4 hrs, or TNFα/AcD for 6 hrs scored for mitochondrial BAX clusters. > 350 cells per condition.

(D) MCF-7_shCON, MCF-7_shRab5A or MCF-7_shRab5C cells, in FM or treated with STS for 4 hrs, or TNFα/AcD for 6 hrs. IF of BAX and cytochrome c. See also Video S8.

(E) Quantifications of cells as in (D) scored for Cyto c– mitochondria. > 600 cells per condition.

(F) Quantifications of cells as in (D) scored for BAX+ mitochondria retaining cytochrome c (BAX+ / Cyto c+). > 600 cells per condition.

(G) Immunoblot of Rab5 levels in HeLa cells stably expressing shCON, shRab5A or shRab5C.

(H) HeLa_shCON, HeLa_shRab5A or HeLa_shRab5C cells, treated with TNFα/AcD +zVAD for 6 hrs. IF of BAX and cytochrome c.

(I) Quantification of cells as in (H), treated with STS for 5 hrs or TNFα/AcD for 6 hrs in the presence of zVAD, scored for Cyto c– mitochondria. > 300 cells per condition.

(J) MCF-7_shRab5C cells, transfected with RFP or RFP-Rab5A, treated with STS for 4 hrs or TNFα/AcD for 6 hrs, scored for Cyto c– mitochondria. > 200 cells per condition.

(K) HeLa_shCON or HeLa_shRab5C, transfected with RFP or RFP-Rab5A, treated with STS or TNFα/AcD in the presence of zVAD. IF of cytochrome c and TOM20, scored for Cyto c– mitochondria. > 160 cells per condition.

(L) MCF-7_Cyto c-GFP cells, transfected with RFP or RFP-Rab5A, treated with STS for 4 hrs, scored for Cyto c– mitochondria. Graph, mean of n = 5 experiments. > 350 cells per condition.

(M) MCF-7_shCON, MCF-7_shRab5A or MCF-7_shRab5C cells, treated with STS for 4 hrs, TNFα/AcD for 6 hrs, or CPT for 24 hrs. IF of Smac and TOM70. See also Figure S3B.

(N) Quantifications of cells as in (J) scored for Smac–mitochondria. > 600 cells per condition.

Nuclei stained with Hoechst. Scale bars, 10 μm. Graphs, mean of n = 3 experiments. Error bars, standard deviation. NS, not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001