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Fig. 4 | Genome Biology

Fig. 4

From: MBD5 and MBD6 stabilize the BAP1 complex and promote BAP1-dependent cancer

Fig. 4

MBD6 but not MBD5 is necessary for tumor cell growth. A The human SCLC cell line NCI-H1963 cells were transduced with either non-targeting CRISPR-Cas9 or two distinct CRISPR-Cas9 sgRNAs of MBD5 and MBD6. The protein levels of MBD5, MBD6, ASXL1-3, and BAP1 were determined by western blot, n=2. B Three different human SCLC cell lines were transduced with either non-targeting CRISPR gRNA, MBD5-specific gRNAs, or MBD6-specific gRNAs for 4 days. The cell numbers were determined by cell counting assay, n=3. Two-tailed unpaired Student’s t test, **P < 0.01; *P < 0.05. C The box plot shows the MBD5 and MBD6 dependency scores of different lung cancer cell types retrieved from the DepMap database. RNA-seq was performed for NCI-H1963 cells transduced with either non-targeting sgRNA, two different MBD5-specific sgRNAs, or two different MBD6-specific sgRNAs. The heatmaps (D) and the MA plot (E) show the differentially expressed genes by the depletion of MBD5 or MBD6, n = 2. F Pathway analysis by Metascape of genes that are downregulated upon MBD6 depletion in NCI-H1963 cells. G The mRNA levels of NEUROG2, DDC, FOXA2, NKX2-8, CDH7, NKX2-1, NXK2-2, and CDK18 genes, which are all critical genes for SCLC development were determined by real-time qPCR in NCI-H1963 cells transduced with non-targeting sgRNA or two different MBD6-specific sgRNAs, n=3. Two-tailed unpaired Student’s t test, **P < 0.01; *P < 0.05. H 1×106 of NCI-H510 SCLC cells transduced with either non-targeting sgRNAs or two distinct MBD6 sgRNAs that were then inoculated into the right flank of athymic nude mice, n=10 per group. Images of representative tumor tissue samples from each mouse were taken at the end of the experiment. I The tumor growth was measured using a calibrated caliper every 2–3 days. Weltch’s t-test was used for statistical analysis. J When each tumor reached 1 cm3, the mouse was euthanized and the survival probability was shown. Log-rank test was performed

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