Fig. 5

Targeting MFRN2 in a MFRN1 deficient background disrupts mitochondrial homeostasis and causes cell death. a Sea horse Mito Stress assay of SNU387 cells targeted with the indicated sgRNA and shRNA combinations. Cells were analyzed for their OCR in a 96-well format before and after the addition of the complex-specific inhibitors. n = 2 biological replicates Left: initial time point, right: 6 days post-DOX treatment. b Immunofluorescence analysis of yH2AX foci by fluorescence microscopy after treatment of SNU387 cells as described in a. Upper panel: Representative images of SNU387 cells with the indicated treatments after 6 days. Primary yH2AX antibody was incubated with Alexa 594-labeled (red) secondary antibody. Nuclear DNA (blue) was counterstained with Hoechst. middle panel: Shown is the mean ± SD of the average yH2AX-foci per cell quantified using ImageJ and counting at least 100 nuclei in 3 biological replicates. Lower panel: Immunoblot analysis of whole cell lysates from SNU387 cells treated as described in a and probed for yH2AX. Vinculin was used as a loading control. Representative results are shown. n = 2 biological replicates. c Cell cycle analysis of SNU387 cells transduced with the indicated plasmid combinations after being cultured in DOX-containing medium for 6 days. FACS analysis was performed at the indicated time points. Results are shown as mean + SD. n = 3 biological replicates. d Apoptosis assay measuring active Caspase-Glo 3/7 by luminescence measurement at the indicated timepoints in SNU387 cells transduced as described above. Shown is the mean ± SD of the relative luminescence compared to CTR cells (sgCTR + shRen). n = 3 biological replicates. e Immunoblot analysis of whole cell lysates from SNU387 cells treated as described above and probed for Caspase-3 and PARP-1. Cleaved protein indicates apoptosis. Vinculin was used as a loading control. Representative results are shown. n = 2 for biological replicates. f Representative images at 40 × magnification showing SA-beta-gal staining of SNU387 cells treated as described above after 6 days of culturing in DOX-containing medium (left panel). Brightfield images were quantified with ImageJ by counting at least 200 cells per condition in 3 independent replicates. Graphs show mean + SD for n = 3 biological replicates. Statistical significance determined by a two-tailed, two-sample t-test (****p < 0.0001, **p < 0.001, *p < 0.05, ns = non-significant) (right panel)