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A. Sarkar et al.
A Figure 4. ATM is localized inside mitochondria: evidence from HeLa cells. (A) Immunoblot analysis (30 mg total protein) of five different stable lentiviral shATM in HeLa cells. A single blot was cut into pieces and probed with the indicated antibodies. Actin was probed as a loading control. (B) Representative confocal z-plane image analysis (scale: 10 mm) showing nuclear and extra-nuclear ATM localization in WT and Kd control (dimethylsulfoxide, DMSO) or CCCP-treated (50 mM for 3 h) HeLa cells. Nuclei are stained with DAPI. (C) Arrows in the inset of Figure 4B showing the local- ization of ATM in the nucleus (N), cytosol (C, green dots) or mitochondria (M, co-local- ized with Tom20, yellow dots). A Laplacian filter was used to identify both ATM and Tom20 co-localization in the merged image. (D) There were significantly fewer cellular ATM dots in whole cells and in all cellular compartments in Kd ATM compared to WT control cells. Significantly more mitochondrial ATM (co-localized ATM-Tom20 dots) was observed in WT HeLa cells treated with CCCP. WT control (162 cells), WT-CCCP (176 cells); Kd control (112 cells) and Kd-CCCP (121 cells) were scanned. Data rep- resent mean ± standard error of mean (n=3 from separate passages of cell lines fol- lowing GFP-Parkin transfection). *P<0.05, ****P<0.0001 significant differences
B from respective controls, as indicated. DAPI represents nuclear staining. (E). Representative confocal z-plane image of WT and Kd HeLa cells showing mitochon- drial nucleoids. Untreated cells were stained with anti-DNA (red) antibody (scale: 10 mm). DAPI represents nuclear staining. (F) Significantly higher (****P<0.0001) mito- chondrial nucleoids in untreated Kd compared to WT HeLa cells. WT (228 cells), and Kd (385 cells) were scanned from three separate passages of cell lines and plotted. (G) WT and Kd HeLa cells were transiently transfected with plasmids (3 mg of each pcDNA control or GFP-Parkin). Representative FCS profile showing GFP expression 36
C
D
EFG
h after transfection (left panel) or merged GFP subsets (right panel).
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haematologica | 2021; 106(2)