Figure 3.

BAF180 knockdown in BAF180-expressing H2 ccRCC cell lines reduces cell survival/proliferation. (a, c, e) Western blot analyses of BAF180 protein in 786-O (a), KC-12 (c) and 769-P (e) H2 ccRCC cells targeted with non-targeting scramble (SCR) shRNA or BAF180 shRNA. (b, d, f) Quantification and photos of the clonogenic survival assay for 786-O (b), KC-12 (d) and 769-P (f) cells targeted with control scr-shRNA or BAF180 shRNA. (g) The location of BAF180 sgRNA #1 and #2 relative to the BAF180-coding region are shown (upper). The diagram (lower) shows the type and frequency of mutations in the BAF180 gene, found in ccRCC tumors, adapted from the article by Valera et al.⁷ (h) DNA sequence and sequencing profiles of parental BAF180 DNA (parental) and BAF180 DNA after targeted by BAF180 sgRNA #1 (left) and #2 (right). The blue lines indicate the target sequences of sgRNAs and the red lines showing the protospacer adjacent motif sequence. The black arrows indicate the positions that double-strand DNA cleavages are expected to occur by the sgRNA-led Cas9 enzyme. The red boxes are to indicate the nucleotides that have been deleted by CRISPR–Cas9 system. SgRNA #2 Clone #1 shows a mixed sequence after the cleavage site, probably due to the fact that the deletion in the BAF180 allele 1 is different from the deletion in BAF180 allele 2. (i) Western blot analysis of BAF180 protein in 786-O/Cas9 cells expressing no sgRNA, or PBRM1 sgRNA #1 (clone #1) or #2 (clone #1). (j) Quantification and photos of the clonogenic survival assay for 786-O/Tet-on Cas9 clones without sgRNA expression, or with expression of BAF180 sgRNA #1 (clone #1) or #2 (clone #1).