Fig. 4

ATF5 directly upregulates DVL1 in bladder cancer cells. A Prediction (upper panel) and validation (lower panel) of potential ATF5-target sites by JASPAR database and ChIP-qPCR assay. B ChIP-qPCR analysis showing enrichment of ATF5 at DVL1 promoter in SW780/UM-UC-3 cells upon ATF5 up-regulation or knock-down. C Luciferase reporter test of DVL1 transcriptional activity upon transfection with ATF5 plasmids and/or luciferase plasmids containing/without DVL1 promoter. D, E qRT-PCR (D) and WB (E) detection of DVL1 expression in ATF5-upregulation or ATF5-knockdown cells compared with control groups. GAPDH was used as the loading control. F WB detection of DVL1 protein in ATF5-overexpressing bladder cancer cells upon transfection with DVL1 siRNAs. G qRT-PCR detection of stemness-related markers (ABCG2, OCT4, SOX2) in ATF5-overepxressing SW780/UM-UC-3 cells upon knockdown of DVL1. H qRT-PCR assay of genes expression of Wnt/β-catenin downstream targets (TCF1, CD44, JUN and CCND1) in ATF5-overepxressing SW780/UM-UC-3 cells transfected with DVL1 siRNAs. I Representative images (left, × 100) and quantitative analyses (right) of tumor sphere formation in ATF5-overexpressig cells upon knockdown of DVL1. Bars represent the mean ± SD of three independent experiments; *P < 0.05, **P < 0.01, ***P < 0.001. qRT-PCR, quantitative real-time polymerase chain reaction; WB, western blotting