Figure 4: KDM3A/B are required to maintain tumorigenic potentials of human colorectal CSCs.

(a) Isolation of ALDH⁺EpCAM⁺ cells from human CRC tissues of patient case #1 (PS1) by FACS. (b,c) The knockdown of KDM3A/B inhibited tumorsphere formation of ALDH⁺EpCAM⁺ cells from PS1. **P<0.01, unpaired two-tailed student’s t-test (n=3). (d) The knockdown of KDM3A/B by siRNA in ALDH⁺EpCAM⁺ cells from PS1. (e) The Topflash activity was significantly higher in ALDH⁺EpCAM⁺ cells than in ALDH⁻EpCAM⁺ cells from PS1. (f) The expression of Wnt target genes was higher in ALDH⁺EpCAM⁺ cells than in ALDH⁻EpCAM⁺. (g) The knockdown of KDM3A/B inhibited the expression of AXIN2, DKK1, CCND1 and MYC in ALDH⁺EpCAM⁺ cells from PS1 (h–j) The knockdown of KDM3A/B significantly inhibited tumour growth of ALDH⁺EpCAM⁺ cells in Nude mice. Tumour growth (i) was monitored for 3 weeks and the weight of the tumors from each group (j) were compared at the end of experiments. **P<0.01, unpaired two-tailed student’s t-test (n=16). (k) The Topflash activity was significantly higher in ALDH⁺HCP1 cells than in ALDH⁻HCP1 cells. (l) The knockdown of KDM3A/B in ALDH⁺HCP1 cells inhibited the expression of AXIN2, DKK1, CCND1 and MYC. (m) The knockdown of KDM3A/B inhibited tumorsphere formation of ALDH⁺HCP1 in vitro. (n) The knockdown of KDM3A/B inhibited the tumorigenic potential of ALDH⁺HCP1 cells in vivo. **P<0.01, unpaired 2-tailed Student’s t-test (n=16).