Fig. 1: Genome-wide CRISPR/Cas9 knockout screen identifies negative regulators of IFNγ-R1 expression to modulate its cell-surface abundance.
From: Ubiquitin ligase STUB1 destabilizes IFNγ-receptor complex to suppress tumor IFNγ signaling
a Spearman correlation of IFNγ-R complex expression with Hallmark IFNγ response signature in scRNA sequencing data37,38,39. SKCM skin cutaneous melanoma, n = 1881; NSCLC non-small-cell lung cancer, n = 5716; BCC, basal cell carcinoma, n = 3551. b Spearman correlation of IFNγ-R complex expression with Hallmark IFNγ response signature in melanoma cell lines treated with MART-1 T cells19, n = 10. c Schematic outline of the FACsorting strategy to establish IFNγ-R1High and IFNγ-R1Low D10 human melanoma cell populations. d Mean Fluorescence Intensity (MFI) of IFNγ-R1 expression on D10 melanoma cells 2 days after sorting the cells as indicated in c. e IFNγ-induced PD-L1 expression of IFNγ-R1High and IFNγ-R1Low cell populations 24 h after IFNγ (10 ng/ml) treatment. f Quantification of the ratio IFNγ-R1High : IFNγ-R1Low in competition assay of (Supplementary Fig. 1e). g Schematic outline of the FACsort-based genome-wide CRISPR-KO screen to identify genes regulating IFNγ-R1 cell-surface expression. h Screen results; red dotted lines indicate FDR cutoff <0.25 for genes enriched in 10% of cells with highest (right) or lowest (left) IFNγ-R1 expression (MAGeCK analysis). Gene names indicate top enriched sgRNAs in cells with the 10% highest IFNγ-R1 expression (right), and sgRNAs targeting IFNGR1 (left), serving as a positive control. i Quantification of IFNγ-R1 expression by flow cytometry on cells expressing the indicated sgRNAs. j IFNγ-R1 expression on D10 melanoma cells measured by flow cytometry in cells expressing indicated sgRNAs. FMO fluorescence minus one, APC Allophycocyanin. k IFNγ-R1 expression (normalized to each respective sgCtrl) measured by flow cytometry in indicated human tumor cell lines expressing either sgCtrl or sgSTUB1. SKCM skin cutaneous melanoma, NSCLC non-small-cell lung cancer, LUAD lung adenocarcinoma, COAD colon adenocarcinoma, THCA thyroid carcinoma, LCML chronic myelogenous leukemia. Mean ± SD in d, e, unpaired t-test for three biological replicates. ****p < 0.0001 (d), ***p = 0.000467 (e). Mean ± SD in f: ****p < 0.0001, ordinary one-way ANOVA for three biological replicates, with Tukey’s post hoc testing. Mean ± SD in i: ****p < 0.0001, ordinary one-way ANOVA for three biological replicates with Dunnett post hoc testing. Mean±SD in k, ****p < 0.0001, multiple t-tests for three biological replicates.
