AML1-ETO–induced cellular functions depend on PLCG1. Proliferation assayed by cell counting after trypan blue exclusion for (A) SKNO-1_Cas9-Blast cells transduced with gRNAs targeting PLCG1, RPA3, or a nontargeting control (gLuc) and (B) Kasumi-1 cells transduced with shRNAs targeting PLCG1or a nontargeting control (shSCR). n = 4-5 independent experiments, 1-way ANOVA. Representative western blot images confirming PLCG1 depletion are shown (day 5 or 7 postinfection). Colony-forming unit analysis in (C) SKNO-1_Cas9-Blast and (D) Kasumi-1 cells on day 14. n = 4-6 independent experiments; paired Student t test. (E) Kaplan-Meier survival curves of humanized NSGS recipient mice, n = 14 mice for shPLCG1-1 or shPLCG1-2 vs n = 10 mice for nontargeting control (shSCR); shown are 3 independent cohorts, Mantel-Cox test. (F-G) Quantitative analysis (left) and representative histograms (right) after flow-cytometric evaluation of CD14 and CD13 expression on (F) Kasumi-1 cells transduced with shRNAs targeting PLCG1or a nontargeting control (shSCR) and (G) SKNO-1_Cas9-Blast cells transduced with gRNAs targeting PLCG1 or a nontargeting control (gLuc). n = 4 independent experiments; paired t test. (H) GSEA of expression changes in 160 hematopoiesis and leukemia-associated gene sets in Kasumi-1 cells transduced with a PLCG1 shRNA (sh1-1) against a nontargeting control (n = 4 for each group). Plotted are normalized enrichment scores (NES) against the log10 false discovery rate (FDR). FDR <0.1 is indicated by the vertical line. (I-J) GSEA showing upregulation of genes bound and repressed by the AML1-ETO fusion protein in Kasumi-1 cells transduced with a PLCG1 shRNA against a nontargeting control. (K) GSEA of expression changes in 160 hematopoiesis and leukemia-associated gene sets in SKNO-1_Cas9-Blast cells transduced with a PLCG1 sgRNA against a nontargeting control (n = 4 for each group). Plotted are normalized enrichment scores (NES) against the log10 FDR. FDR <0.1 is indicated by the vertical line.