Extended Data Fig. 9: IRF1 K78 acetylation promotes the DNA binding of IRF1.
a, ChIP-qPCR analysis of ectopically expressed IRF1-3MYC or IRF1 K78R-3MYC in HCT116 cells. Data are shown as the mean ± SD, n = 3 biologically independent experiments. p values were calculated by two-tailed Student’s t-test. b, HEK293T cells were transiently cotransfected with the indicated plasmids and analyzed by immunoprecipitation. c-e, Schematics of the locus-specific IRF1 K78R knock-in A375 cell line constructed by CRISPR-Cas9-mediated homology-directed repair. See Methods for details. An sgRNA efficiently targeting IRF1 was used (c). A DNA donor template containing internal ribosome entry site (IRES), EGFP, and indicated mutations sequence in the intron sequence between exon 3 and exon 4 was used (d). Site-specific PCR amplification and Sanger sequencing of the K78R gene-edited clone (e). f, qRT-PCR analysis of PD-L1 mRNA levels in parental WT and K78R A375 cells. Data are shown as the mean ± SD, n = 3 biologically independent experiments. p values were calculated by two-tailed Student’s t-test. g,h Representative confocal images showing FRAP of the puncta formed by mEGFP-KAT8 and IRF1-mCherry or IRF1 K78R-mCherry in 143B cells (g). FRAP curves are shown on the right (h). The data are plotted as the mean ± SD (n = 5 biologically independent experiments). The p value was calculated by two-tailed Student’s t-test. i, HEK293T cells were transiently cotransfected with indicated constructs and analyzed by immunoprecipitation. The experiments in b,i were repeated three times with similar results.
