KDM5 histone demethylases repress immune response via suppression of STING
Fig 6
KDM5 inhibition induces a robust interferon response in cancer cells with elevated levels of cytosolic DNA.
(A) Immunostaining of dsDNA and the mitochondrial marker Hsp60 in MCF7 cells. Surface plots of Z-stack images generated with Huygens (left panel). qPCR detecting mtDNA copy number in cells with the indicated treatment (right panel). 10 μM of ddC was used. Scale bar, 10 μm. (B, C) RT-qPCR (panel B) and western blot (panel C) analyses of MCF7 cells with the indicated treatment. MCF7 cells were treated with 10 μM ddC and 1 μM KDM5-C70 for 6 days. Cells were refed every 2 days. (D) dsDNA and DAPI staining of MCF7 cells or SKBR3 cells digested with 50 μg/ml DNase I. Surface plots of Z-stack images generated with Huygens (left panel). Quantification of dsDNA intensity per cell using image J was shown in the right panel. Scale bar, 10 μm. (E) RT-qPCR analyses of SKBR3 cells with the indicated treatment. SKBR3 cells were transfected with 1 μg dsDNA of about 90 bp (dsDNA90) using Lipofectamine 2000 5 hours before treatment with DMSO or 1 μM KDM5-C70 for 3 days. (F) RT-qPCR analysis of SKBR3 cells with knockout of the indicated genes after treatment with 1 μg dsDNA and 1 μM KDM5-C70 for 3 days. Representative data from triplicate experiments are shown. Error bar denotes SEM. #p < 0.01 for panel A, B, and D–F, for KDM5-C70 + dsDNA90 versus DMSO + lipo (panel E), and for knockout sgRNA versus control sgRNA (panel F). The numerical values used to generate graphs in panel A, B, and D–F are available in S1 Data. ddc, dideoxycytidine; dsDNA, double-stranded DNA; lipo, Lipofectamine 2000; mtDNA, mitochondrial DNA; RT-qPCR, RT-qPCR, reverse transcription followed by quantitative PCR; sgRNA, single guide RNA.