Figure S4. Knockout or knockdown of Rab... | Rockefeller University Press

Figure S4.

$Knockout or knockdown of Rab GTPase impaired mitophagy. (A and B) HEK293T cells transiently expressing shRNA of Rab8 (Rab8 KD) or HA-GFP-Rab8 (Rab8 OE), cells were treated with DMSO or OA (10 mM oligomycin, 4 mM antimycin A) for 0, 18, or 24 h, respectively. The endogenous Rab8 or COXII were measured by western blot. Quantification is shown in B (n = 3 experimental replicates). Data are shown as means ± SEM and analyzed with one-way ANOVA. ***P < 0.001. (C and D) Rab8 KO HeLa cells transiently expressing HA-Vector, HA-Rab8, HA-Rab8 Q67L, and HA-Rab8 T22N, 24 h after transfection, were treated by 10 µM CCCP for 0, 2, or 4 h, whole cell lysates were collected and the levels of endogenous COXII was measured by western blot. Quantification is shown in D (n = 3 experimental replicates). Data are shown as means ± SEM and analyzed with one-way ANOVA. ***P < 0.001. (E) GFP, OM45-GFP-atg5∆, OM45-GFP-Ypt1KD, and OM45-GFP-Ypt7∆yeast strains were cultured in SD-N medium for 0, 4, and 8 h. The cleavage of OM45-GFP was analyzed by western blot. Pgk1 served as a loading control. (F) OM45-GFP, OM45-GFP-atg5∆, OM45-GFP-Ypt31∆, and OM45-GFP-Yp32∆yeast strains were cultured in SD-N medium for 0, 4, and 8 h. The cleavage of OM45-GFP was analyzed by western blot. Pgk1 served as a loading control. (G) OM45-GFP, OM45-GFP-atg5∆, OM45-GFP-Ypt51∆, and OM45-GFP-Yp52∆yeast strains were cultured in SD-N medium for 0, 4, and 8 h. The cleavage of OM45-GFP was analyzed by western blot. Pgk1 served as a loading control. (H) OM45-GFP, OM45-GFP-atg5∆, OM45-GFP-Ypt7∆, and OM45-GFP-Ypt7∆ (FLAG-Ypt7) yeast strains were cultured in SD-N medium for 0, 4, 8 h. The cleavage of OM45-GFP was analyzed by western blot. Pgk1 served as a loading control. (I) Representative confocal images of Pcol-19-mRFP::GFP::FIS1(zjuSi374) transgenic animals treated with rab-1, rab-2, rab-7, rab-8, rab-11.1, rab-21, rab-39, and control L4440 (empty vector) RNAi for 4 h at DMSO treatment. Scale bars, 10 µm. The scale bars in the magnification boxes are 2 µm. (J) PINK1 WT and PINK1 KO HEK293 cells were treated with 10 µM CCCP or 10 µM CCCP and Bafilomycin A1 for 2 h, and fractions were isolated. The Input group represents the total proteins, the Cyto group represents the cytoplasmic component, and the Mito group represents the mitochondrial component proteins, which were collected and analyzed by western blot. (K) Atg7 KO U2OS cells transiently expressing HA-ULK1, GFP-Rab8, and mCherry-NDP52 were treated with DMSO or CCCP for 4 h, the colocalization of Rab8, ULK1, NDP52, and endogenous TOMM20 were analyzed by confocal microscopy. Scale bars, 10 µm. The scale bars in the magnification boxes are 2 µm. (L) Atg7 KO U2OS cells transiently expressing GFP-Rab8 and mCherry-NDP52, were treated with DMSO or CCCP for 4 h, the colocalization of Rab8, ATG9, NDP52, and endogenous TOMM20 was analyzed by confocal microscopy. Scale bars, 10 µm. The scale bars in the magnification boxes are 2 µm. (M) Rab8 WT U2OS cells transiently expressing GFP-Rab8 and mCherry-NDP52, were treated with DMSO or CCCP for 4 h, the colocalization of GFP-Rab8, ATG9, NDP52, and endogenous TOMM20 was analyzed by confocal microscopy. Scale bars, 10 µm. The scale bars in the magnification boxes are 2 µm. (N) Rab8 KO U2OS cells transiently expressing GFP-C1 and mCherry-NDP52, were treated with DMSO or CCCP for 4 h, the colocalization of GFP-C1, ATG9, NDP52, and endogenous TOMM20 was analyzed by confocal microscopy. Scale bars, 10 µm. The scale bars in the magnification boxes are 2 µm. Molecular weight measurements are in kD. Source data are available for this figure: SourceData FS4.$

Knockout or knockdown of Rab GTPase impaired mitophagy. (A and B) HEK293T cells transiently expressing shRNA of Rab8 (Rab8 KD) or HA-GFP-Rab8 (Rab8 OE), cells were treated with DMSO or OA (10 mM oligomycin, 4 mM antimycin A) for 0, 18, or 24 h, respectively. The endogenous Rab8 or COXII were measured by western blot. Quantification is shown in B (n = 3 experimental replicates). Data are shown as means ± SEM and analyzed with one-way ANOVA. ***P < 0.001. (C and D) Rab8 KO HeLa cells transiently expressing HA-Vector, HA-Rab8, HA-Rab8 Q67L, and HA-Rab8 T22N, 24 h after transfection, were treated by 10 µM CCCP for 0, 2, or 4 h, whole cell lysates were collected and the levels of endogenous COXII was measured by western blot. Quantification is shown in D (n = 3 experimental replicates). Data are shown as means ± SEM and analyzed with one-way ANOVA. ***P < 0.001. (E) GFP, OM45-GFP-atg5∆, OM45-GFP-Ypt1KD, and OM45-GFP-Ypt7∆yeast strains were cultured in SD-N medium for 0, 4, and 8 h. The cleavage of OM45-GFP was analyzed by western blot. Pgk1 served as a loading control. (F) OM45-GFP, OM45-GFP-atg5∆, OM45-GFP-Ypt31∆, and OM45-GFP-Yp32∆yeast strains were cultured in SD-N medium for 0, 4, and 8 h. The cleavage of OM45-GFP was analyzed by western blot. Pgk1 served as a loading control. (G) OM45-GFP, OM45-GFP-atg5∆, OM45-GFP-Ypt51∆, and OM45-GFP-Yp52∆yeast strains were cultured in SD-N medium for 0, 4, and 8 h. The cleavage of OM45-GFP was analyzed by western blot. Pgk1 served as a loading control. (H) OM45-GFP, OM45-GFP-atg5∆, OM45-GFP-Ypt7∆, and OM45-GFP-Ypt7∆ (FLAG-Ypt7) yeast strains were cultured in SD-N medium for 0, 4, 8 h. The cleavage of OM45-GFP was analyzed by western blot. Pgk1 served as a loading control. (I) Representative confocal images of Pcol-19-mRFP::GFP::FIS1(zjuSi374) transgenic animals treated with rab-1, rab-2, rab-7, rab-8, rab-11.1, rab-21, rab-39, and control L4440 (empty vector) RNAi for 4 h at DMSO treatment. Scale bars, 10 µm. The scale bars in the magnification boxes are 2 µm. (J) PINK1 WT and PINK1 KO HEK293 cells were treated with 10 µM CCCP or 10 µM CCCP and Bafilomycin A1 for 2 h, and fractions were isolated. The Input group represents the total proteins, the Cyto group represents the cytoplasmic component, and the Mito group represents the mitochondrial component proteins, which were collected and analyzed by western blot. (K) Atg7 KO U2OS cells transiently expressing HA-ULK1, GFP-Rab8, and mCherry-NDP52 were treated with DMSO or CCCP for 4 h, the colocalization of Rab8, ULK1, NDP52, and endogenous TOMM20 were analyzed by confocal microscopy. Scale bars, 10 µm. The scale bars in the magnification boxes are 2 µm. (L) Atg7 KO U2OS cells transiently expressing GFP-Rab8 and mCherry-NDP52, were treated with DMSO or CCCP for 4 h, the colocalization of Rab8, ATG9, NDP52, and endogenous TOMM20 was analyzed by confocal microscopy. Scale bars, 10 µm. The scale bars in the magnification boxes are 2 µm. (M) Rab8 WT U2OS cells transiently expressing GFP-Rab8 and mCherry-NDP52, were treated with DMSO or CCCP for 4 h, the colocalization of GFP-Rab8, ATG9, NDP52, and endogenous TOMM20 was analyzed by confocal microscopy. Scale bars, 10 µm. The scale bars in the magnification boxes are 2 µm. (N) Rab8 KO U2OS cells transiently expressing GFP-C1 and mCherry-NDP52, were treated with DMSO or CCCP for 4 h, the colocalization of GFP-C1, ATG9, NDP52, and endogenous TOMM20 was analyzed by confocal microscopy. Scale bars, 10 µm. The scale bars in the magnification boxes are 2 µm. Molecular weight measurements are in kD. Source data are available for this figure: SourceData FS4.