Fig. 5

YAP1 inhibits miR-124-3p and miR-188-5p expression by recruiting YY1 to the promoters. A, B RT-qPCR analysis of the mRNA levels of DCLK1, Vimentin and concurrent expression levels of miR-124-3p and miR-188-5p in HCT116 and SW620 cells transfected with YY1 siRNAs. C Western blot analysis of the expression of YY1, DCLK1 or Vimentin in HCT116 and SW620 cells transfected with two different YY1 siRNAs. β-actin was used as an internal control. D Western blot analysis of YY1, DCLK1 or Vimentin in HCT116 and SW620 cells transfected with YY1 siRNA and then rescued with HA-YY1 plasmid. E RT-qPCR analysis of miR-124-3p and miR-188-5p levels in HCT116 cells transfected with YY1 siRNA and HA-YY1 plasmid compared with siNC and vector control respectively. Bars are means ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, n = 3. F Analysis of the interaction of endogenous YAP1 with YY1 and transfected Flag-YAP-5SA with HA-YY1 by Co-IP. IP was performed using anti-YAP1 or anti-Flag antibodies and normal rabbit IgG antibodies. G IF staining was performed to determine the colocalization of YAP1 (red) and YY1 (green) in HCT116 and HT29 cells treated with CXCL12 (100 ng/ml) in the presence of AMD3100 (2 μM). DAPI was used for nuclear staining. Scale bars, 50 µm. (H) HCT116 cells were co-transfected with miR-124-3p or miR-188-5p promoter luciferase construct (miR-124 or miR-188 promoter) together with HA-YY1 plasmid. pGL3-basic plasmid was used as the vector control. The comparison of luciferase activities of promoter constructs normalized to Renilla activity was indicated. Bars are means ± SD; *P < 0.05, **P < 0.01 (n = 3)