Fig 2: A sensitive and efficient inducible CRISPR/Cas9 system. (A) Schematic representation of the Lenti-iCas9-neo plasmid. LTR, long terminal repeat; PSI, retroviral ? packaging element; RRE, Rev response element; TRE2, TRE2 promoter; P2A, a 2A self-cleavage peptide from porcine teschovirus-1; Ubc, Ubiquitin C promoter; rtTA3, reverse tetracycline-controlled transactivator 3; IRES, internal ribosome entry site; Neo, neomycin resistance. (B) The indicated cells were transduced with Lenti-iCas9-neo and LentiGuide carrying the indicated sgRNA. Cells were then treated with or without 1 µg/ml doxycycline (DOX) for 72 h and analyzed by western blots. (C) Overview of the FACS sorting strategy. (D) Unsorted and sorted HeLa/iCas9 cells were treated with or without 1 µg/ml DOX for 24 h and GFP expression was analyzed by FACS. (E–G) SKBR3 (E), Hela (F) and MCF10A (G) iCas9 cells before or after FACS sorting were transduced with LentiGuide carrying control or KDM5C sgRNA. Cells were then treated with or without 1 µg/ml DOX for 72 h and analyzed by western blots.

Fig 3: IFN-responsive genes were downregulated by VHL and upregulated by KDM5C.(A) Venn diagram showing that KDM5C loss had greater impact on transcriptome in VHL-/- cells than that in VHL+/+ cells; (B) Venn diagram showing the overlap of genes downregulated by VHL and upregulated by KDM5C in 786-O cells. P-values were calculated with hypogeometric probability. (C) Enriched gene ontology (GO) analysis of genes regulated both by VHL and KDM5C. (D) GSEA analysis of genes upregulated by KDM5C. (E) Heatmap showing the expression of IFN-responsive genes after KDM5C knockdown.

Fig 4: HIF, PBRM1 and KDM5C are required for the expression and activity of ISGF3.A Venn diagram showing the overlaps of genes whose expression was affected by VHL, PBRM1 or KDM5C losses in 786-O cells. The p-value significance of the overlap in a venn diagram was calculated using hypergeometric test in R; (B) Heat map of shared gene targets by VHL, PBRM1 and KDM5C; (C) Enriched gene ontology (GO) analysis of genes regulated both by VHL, PBRM1 and KDM5C; (D) Western blots with the indicated antibodies in 786-O cell SDS-solubilized whole cell lysates with HIF2a, PBRM1 or KDM5C suppressed by shRNA; (E) Examination of mRNA expression of individual genes by RT-qPCR in 786-O cells after PBRM1 or KDM5C suppression by shRNA; (F) Western blots with the indicated antibodies in Ren-02 cell SDS-solubilized whole cell lysates with HIF2a, PBRM1 or KDM5C suppressed by shRNA. The p-value significance in the bar graph was calculated with two-tailed student t test. *p<0.05; **p<0.01; ***p<0.001.

Fig 5: Tumor suppressors could be potential anticancer targets in certain cancers.(A) Relative essentiality of TSC2 and mTOR in 293A WT and TSG KO cells as shown by QBF. Light red dashed line indicates the range of the mean QBF_TSC2 from all four WT screens ± 3 × SD. Black dashed line indicates the range of the mean QBF_mTOR from all four WT screens ± 3 × SD. (B) gRNA fold changes of TSC2 in 293A WT, LKB1 KO, and PTEN KO cells. (C) KDM5C gene dependency in 293A WT and TSG KO cells as shown by QBF. Light green dashed line indicates the range of the mean QBF_KDM5C from all four WT screens ± 4 × SD. (D) KDM5C KO effects inferred by the CERES dependency scores were compared in 67 breast and kidney cancer cell lines grouped by BAP1 mutation status using data from the DepMap portal. Data are presented as means ± SEM and analyzed with an unpaired t test. *P < 0.05. (E) Oncoprint displaying mutational landscapes of LKB1 and TSC2 in patients with non–small cell lung cancer on cBioPortal. Copy number alterations were not included in this analysis. Alteration colors are the same as those shown in cBioPortal. Data were retrieved on 1 August 2021. (F) Oncoprint displaying mutational landscapes of BAP1 and KDM5C in patients with ccRCC on cBioPortal. Other conditions are the same as in (E).