Fig. 1: CRISPR–Cas9 screening identified KAT8 as a PD-L1 regulator.
a, Schematic of the experimental design. b, Plot of whole-genome CRISPR–Cas9 gene knockout screen results using MAGeCK analysis. Cells were sorted after 14 d of infection. The x axis indicates the fold change of each gene, the y axis shows the Robust Rank Aggregation (RRA) score of each gene, and the bubble size of the indicated genes indicates the number of good sgRNAs. c,d, Western blotting (c) and quantitative PCR with reverse transcription (RT–qPCR) (d) analyses of A375 cells expressing the indicated sgRNAs in the presence or absence of 100 U ml–1 IFNγ for 6 h; n = 3 biologically independent experiments. e,f, A375 cells expressing the indicated sgRNAs were infected with sgRNA-resistant WT KAT8 or the C316S mutant, as indicated, and analyzed by western blotting (e) and RT–qPCR (f); n = 3 biologically independent experiments. g, Cytotoxicity assay of A375 cells expressing the indicated sgRNAs; n = 3 biologically independent experiments. h–m, LLC1 cells expressing the indicated sgRNAs were subcutaneously injected into mice. Tumor growth (h) and tumor weights (i) were measured, and the extent of CD3+CD8+ T cell infiltration was analyzed by FACS (j). Tumor slices were stained with anti-PD-L1 and anti-CD8 (k), and CD8+ cell counts (l) and mean fluorescence intensity (MFI) of PD-L1 (m) were analyzed. Data in h–m were generated from n = 6 mice for each group. n,o, Mice bearing tumors formed by LLC1 cells expressing sgNC and sgKAT8 were treated with IgG or anti-PD-1 on days 4, 6 and 8. Tumor sizes were measured at the indicated time points (n). Weights of the resected tumors were measured at the endpoint (o); n = 6 mice per group. Error bars in d, f–j and l–o indicate the mean ± s.d. P values in h and n were calculated by two-way ANOVA with Tukey’s multiple-comparison test. P values in d, f, g, i, j, l, m and o were calculated by two-tailed Student’s t-test. p, Crosstab shows the distribution of cancer tissues in the human multiple organ cancer tissue arrays according to the median IHC score of PD-L1 and KAT8. The P value and chi-square value were calculated using Pearson’s chi-square test, and the R value was calculated using Spearman’s correlation test.
