Journal: eLife

Article Title: Unleashing a novel function of Endonuclease G in mitochondrial genome instability

doi: 10.7554/eLife.69916

Figure Lengend Snippet: ( A ) Localization of Endonuclease G in mitochondria in different cell lines. Representative images of localization of Endonuclease G to mitochondria in HeLa, MEF and HEK293T cells. FITC-conjugated secondary antibodies were used for detecting Endonuclease G proteins. MtDR is Mitotracker Red, a mitochondrial marker. DAPI is used as nuclear stain. ( B ) Colocalization analyses of Endonuclease G and Mitotracker signals using JaCoP in ImageJ software based on immunofluorescence studies performed in multiple cell lines (see ). Minimum of 50 cells were used for analysis of colocalization of red and green signals and plotted as the colocalization value of green overlapping red. Y- axis depicts the Mander’s colocalization coefficient value calculated for green over red and plotted in the form of dot plot. The significance was calculated using GraphPad Prism 5.0 with respect to secondary control and shown as mean ± SEM ( ns not significant, *p<0.05, **p<0.005, ***p<0.0001). ( C ) Representative images of colocalization of Endonuclease G to mitochondrial matrix (TFAM) in HeLa cells. Conjugated secondary antibodies were used for detecting Endonuclease G (Alexa Fluor 488) and mitochondrial matrix protein, TFAM (Alexa Fluor 568). DAPI is used as nuclear stain. ( D ) Colocalization analyses of Endonuclease G and TFAM signals using JaCoP in ImageJ software. Minimum of 50 cells were used for analysis of colocalization of red and green signals and plotted. Y-axis depicts the Mander’s colocalization coefficient value calculated for green over red and plotted in the form of dot plot. Control represents the panel where only one of the primary antibodies was used. ( E ) Knockdown of Endonuclease G from HeLa cell using PEI mediated transfection. shRNA against Endonuclease G cloned plasmid was used for transfection. Cells were harvested after 48 h and mitochondrial extracts were prepared. Western blotting was performed to confirm the knockdown of Endonuclease G from the HeLa cells. Actin served as a loading control. ( F ) The knockdown extract was incubated with the plasmid and used for the primer extension using VKK11 primer (‘I’ and ‘II’ are two biological repeats). Extract prepared from the sample transfected with scrambled plasmid served as a control (SCR control). Lanes 3, 4, 7, and 8 serve as scrambled controls while lanes 5, 6, 9, and 10 are for knockdown extracts. I and II represent two independent batches of experiments. ‘M’ is a 50 nt ladder. ( G ) Bar diagram representing the cleavage intensity of the extracts prepared after transfection with scrambled plasmid and shEndo G plasmid. In panels F and G, a minimum of three biological repeats were performed and the data is shown with the error bar calculated as SEM (ns: not significant, *p<0.05, **p<0.005, ***p<0.0001). Refer also . Figure 8—source data 1. Localization of Endonuclease G to mitochondria. Figure 8—source data 2. Immunofluorescence showing the Localization of Endonuclease G to mitochondria.

Article Snippet: HeLa (human cervical cancer), HCT116 (human colon cancer), MEF (mouse embryonic fibroblast) and HEK 293T (human embryonic kidney epithelial cell line) were purchased from National Centre for Cell Science, Pune, India.

Techniques: Marker, Staining, Software, Immunofluorescence, Control, Knockdown, Transfection, shRNA, Clone Assay, Plasmid Preparation, Western Blot, Incubation