Splicing junction site mutations of PKM gene disrupt endogenous isoform‐specific gene splicing. a‐b. Schematic diagram of establishment of E9‐GT A, and E10‐GT B, mutant cell lines from single clones. The E9‐GT mutant cells were diluted and plated onto 96‐well plates for culture. After 10 d, the single clone‐derived cell lines were subjected to Sanger sequencing for genotyping. Three single clone‐derived cell lines were established for E9‐GT (Mut1‐3) and E10‐GT (Mut4‐6) mutant HCT116 cells. C, Detection of PKM1 and PKM2 expression by gel electrophoresis with expected length of PCR fragments in wild‐type (WT, WT2 and WT3), E9‐GT (Mut1‐3) and E10‐GT (Mut4‐6) mutant HCT116 cell lines. D, Semi‐quantitative analysis of PKM1 and PKM2 expression relative to GAPDH in c using ImageJ software. E‐F. Total RNA from wild‐type (WT, WT2 and WT3), E9‐GT (Mut1‐3) and E10‐GT (Mut4‐6) mutant HCT116 cell lines was subjected to RNA‐seq analysis. The enrichment of transcripts within exons or introns was presented by IGV viewer