Extended Data Fig. 7: TFAM deficiency induces mtDNA stress and DNA-damage resistance in MC-38 mouse colon cancer cells.
From: Mitochondrial DNA stress signalling protects the nuclear genome
a-e, WT and TFAM deficient (TFD) MC-38 cells (see Methods section) were analysed. a, RT-qPCR analysis of Tfam mRNA. b, western blot probing STAT1 (L.E., long exposure; S.E., short exposure), TFAM and GAPDH (loading control) (n=3 independent experiments). c, mtDNA abundance (relative mtDNA copy number) by qPCR with D-loop primers. d, mitochondrial and mtDNA nucleoid morphology analyses by immunofluorescence against HSP60 (Mito., magenta) and DNA (DNA, cyan) (n=3 independent experiments). Images are Z-stack projections and scale bar represents 10 µm. e, RT-qPCR analysis of the indicated ISGs. f, Western blot analysis in WT and TFD MC-38 cell pools transduced with the indicated gene-specific guide RNA (gRNA) or scrambled (Scr) control gRNA to determine the knockout efficiency (n=1, only to validate). GAPDH was probed as the loading control. g, RT-qPCR analysis of the indicated ISGs in WT and TFD MC-38 cell pools transduced with the indicated gene-specific gRNAs (described in f). h, Cell survival analysis of WT and TFD MC-38 cells challenged with 500 nM doxorubicin for 48 h using the alamarBlue assay (n=7 biological replicates). a, c, e and g, the data shown are from one of three biological replicates with the error bars indicating the mean ± s.d. of three technical replicates. The other two biological replicates are provided as Supplementary Fig. 9. All data were analysed with two-tailed unpaired student’s t tests. Asterisks indicate significance as follows:. ** P < 0.01, *** P < 0.001, **** P < 0.0001.
