Fig. 1

MCCC2 promoted cell proliferation, invasion, and migration in vitro. A Western blotting confirmed the knockdown efficiency in HCT116 stable cell line transfected with MCCC2 shRNA or control plasmid. B Western blotting confirmed the overexpression of MCCC2 in HCT116 stable cell line transfected with FLAG-MCCC2 or control plasmid. Arrows indicated the exogenous and endogenous form of MCCC2 protein, respectively. C Three sgRNAs targeting sites were locating in exon 1 of MCCC2 gene (ATG being the initiation codon). D After transfection, and selection, one particular single clone was identified by Sanger sequencing to harbor homozygous 56 bp deletion plus 1 bp insertion in exon 1 of MCCC2 gene, resulting in premature termination codon. E Western blotting to confirm the knock out (KO) of MCCC2 gene in D). F–H Proliferation assay by RTCA in pairwise cell lines showed that MCCC2 selective expression could affect cell proliferation. Two‐way ANOVA was used to calculate p value. I–K Representative images and quantification of colony formation assay in pairwise cell lines showed that MCCC2 selective expression could affect colony formation ability (n = 3). Paired two-tailed Student’s t-test was used to calculate p value. L–N Representative images and quantification of wound healing in pairwise cell lines showed MCCC2 selective expression could affect the cellular migration ability (n = 3). Paired two-tailed Student’s t-test was used to calculate p value. O–Q Representative images and quantification of Transwell assay in pairwise cell lines showed that MCCC2 selective expression could affect the cellular invasion ability (n = 3). Paired two-tailed Student’s t-test was used to calculate p value. R Proliferation assay by IncuCyte showed that after re-expression of MCCC2 in the KO cells enhanced cell proliferation. Two‐way ANOVA was used to calculate p value. S Representative images and quantification of colony formation assay showed that re-expression of MCCC2 in the KO cells enhanced colony formation ability (n = 3). Paired two-tailed Student’s t-test was used to calculate p value. (All data represent the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)