Fig. 3.

C9orf140 is a negative regulator of Wnt/β-catenin signaling. a The 293T cells were transfected with C9orf140, Axin1, or empty vector, together with TCF optimal (TOP) or mutant negative control (FOP) luciferase reporter, TK-Renilla reporter and Wnt1. The firefly luciferase activity measured by using the Dual-Luciferase Reporter Assay System was normalized to the Renilla luciferase activity. Data are presented as average fold activation relative to empty vector-treated cells +SD in the FOP-transfected or TOP-transfected group. b TOPFlash luciferase assays were performed in 293T cells transfected with C9orf140, Axin1, or empty vector. Luciferase activities were measured after treatment with control L cell-conditioned medium (L-CM) or Wnt3A-conditioned medium (Wnt3A-CM) for 18 h. Data are normalized to empty vector-transfected cells and presented as the mean + SD. c Real-time PCR for AXIN2, MYC, CCND1 and CTNNB1, normalized to GAPDH expression, in 293T cells transfected and treated as in (b). The ratio of normalized gene expression in Wnt3A-CM-treated cells relative to the L-CM-treated cells, in empty vector-transfected cells, was set to 1. The values of other groups were normalized to this value and are presented as the mean + SD. d RKO cells were transfected with C9orf140, Axin1, or empty vector, together with TOP or FOP luciferase reporter and TK-Renilla reporter. Luciferase activities were measured after treatment with L-CM or Wnt3A-CM for 18 h. Data are presented as average fold activation relative to empty vector-treated cells + SD in FOP-transfected or TOP-transfected group. e TOPFlash luciferase assays were performed in control 293T cells or two C9orf140 knockout cell lines. Luciferase activities were measured after treatment with L-CM or Wnt3A-CM for 18 h. Data are normalized to L-CM-treated control cells and presented as the mean + SD. The upper panel shows western blot verification of endogenous C9orf140 knockout by CRISPR/Cas9 in 293T cells. All results in the figure are representative of at least three experiments performed in duplicate. Statistical significance was calculated by one-way ANOVA, and significance was defined as ***p < 0.001, **p < 0.01, or *p < 0.05