Fig 2: Molecular mechanism underlying ZNF575 inhibiting CRC growth ZNF575 promotes p53 expression and activates p53 signaling pathway via directly targeting p53 promoter, hence promotes BAK and PUMA transcription.

Fig 3: p53 plays a necessary role in ZNF575 inhibiting CRC proliferation. (A) Detection of p53 protein expression in HCT116-Control, HCT116-ZNF575, RKO-Control and RKO-ZNF575 cells that were transfected with shRNA and shp53 by Western blotting. (B and C) HCT116-Control, HCT116-ZNF575, RKO-Control and RKO-ZNF575 cells that were transfected with shRNA and shp53 were seeded in 96-well plate (1,000 cells/well). The cell activity was measured by CCK-8 kit at 72 h post cell seeding. The relative cell activity was analyzed (n = 4, **p < 0.01). (D and E) HCT116-Control, HCT116-ZNF575, RKO-Control and RKO-ZNF575 cells that were transfected with shRNA and shp53 were seeded in 6-well plate (2,000 cells/well). Ten days post cell seeding, the colony was stained and analyzed (n = 3, **p < 0.01; ns, no significant difference).

Fig 4: Ectopic expression of ZNF575 inhibits CRC cell proliferation. (A) Determination of ZNF575 mRNA expression in HCoEpiC and several CRC cell lines (DLD-1, HT-29, RKO, SW480, HCT116, SW620) by qPCR (n = 3, **p < 0.01, compared to HCoEpiC). (B) Determination of ZNF575 protein expression in HCoEpiC and several CRC cell lines (DLD-1, HT-29, RKO, SW480, HCT116, SW620) by qPCR. (C) Determination of ZNF575 protein expression in HCT116-Control (Ctrl), HCT116-ZNF575, RKO-Control and RKO-ZNF575 cells. (D and E) HCT116-Control, HCT116-ZNF575, RKO-Control and RKO-ZNF575 cells were seeded in 96-well plate (1,000 cells/well). The cell activity was measured by CCK-8 kit (n = 4, **p < 0.01). (F and G) HCT116-ControlControl, HCT116-ZNF575, RKO-ControlControl and RKO-ZNF575 cells were seeded in 6-well plate (2,000 cells/well). Ten days post cell seeding, the colony was stained by crystal violet. The number of colonies was counted and analyzed (n = 3, **p < 0.01). (H and I) HCT116-Control, HCT116-ZNF575, RKO-Control and RKO-ZNF575 cells were collected for apoptotic cells detection by flowy cytometry. The apoptotic percentage in each group was analyzed (n = 3, **p < 0.01).

Fig 5: Ectopic expression of ZNF575 impairs CRC tumor growth in mice, (A) Detection of ZNF575 expression in HCT116-Control and HCT116-ZNF575 tumors by IHC staining (Scale bar = 100 μm). The percent of ZNF575 positive cells in HCT116-Control tumors and HCT116-ZNF575 tumors was analyzed (n = 3, **p < 0.01). (B) Image of HCT116-Control and HCT116-ZNF575 tumors at 20 days post cell injection. (C) Tumor growth curve of HCT116-Control and HCT116-ZNF575 tumors (n = 5, **p < 0.01). (D) Tumor weight of HCT116-Control and HCT116-ZNF575 tumors (n = 5, **p < 0.01). (E) Image of RKO-Control and RKO-ZNF575 tumors at 20 days post cell injection. (F) Tumor growth curve of RKO-Control and RKO-ZNF575 tumors (n = 5, **p < 0.01). (G) Tumor weight of RKO-Control and RKO-ZNF575 tumors (n = 5, **p < 0.01). (H) Detection of apoptotic cells in HCT116-Control and HCT116-ZNF575 tumors by TUNEL assay (Scale bar = 200 μm). The number of apoptotic cells in HCT116-Control tumors and HCT116-ZNF575 tumors was analyzed (n = 3, **p < 0.01).