Figure 4: BRG1-embedded BAF complex initiates YAP1 signalling in liver CSCs.

(a) BRG1 and BRM KO cells were established by CRISPR/Cas9 approaches, followed by examination of main self-renewal pathway target genes. Gene fold changes were determined by RT–qPCR. CRISPR/Cas9 caused frameshift mutations with no changes in mRNA levels. (b) YAP1 targets were tested by immunoblotting in BRM KO spheres (left panel) or BRG1 KO spheres (right panel). (c) Schematic diagram of YAP1 promoter (left panel) and domain mapping (right panel). HCC primary spheres were collected for ChIP assay with BRG1, BRM antibodies. TSS, transcription start site. (d) The binding region of YAP1 promoter to BRG1 was validated by luciferase assay. (e) Biotin-labelled YAP1 promoter region (−420∼−380 bp) was used for EMSA assay. BRG1 was immunoprecipitated from Huh7 spheres using BRG1-specific antibody. (f) Oncosphere nuclear lysates of the indicated cells were treated with DNase I, followed by real-time PCR. (g) BRG1 KO or BRM KO spheres were used for ChIP assays using H3K4me3 antibody. (h) BRG1-binding region of YAP1 promoter was deleted in HCC primary tumour cells using CRISPR/Cas9 technology, followed by depletion of BRG1 or BRM. Total RNA was extracted for PCR assay. YAP1PKO, YAP1 promoter KO. Data are shown as means±s.d. Two tailed Student’s t-test was used for statistical analysis; *P<0.05 and **P<0.01; NS, not significant. Data are representative of three independent experiments.