Figure 3. CHIR enhances proliferation of primary bladder cancer cells in organoid culture with activation of Wnt/β-catenin pathway.

(A) Representative images of primary cell organoids (BCa #01) at day 1 and day 6 treated with DMSO or CHIR of 10 µM. Scale bar, 100 µm. (B) Relative growth of organoids from 4 patient samples was calculated by dividing area at day 6 by that at day 1, and depicted in the plot. (C) Relative viability of organoids from 2 patient samples was measured by dividing the amount of ATP at day 6 by the area of organoid at day 1, and depicted in the plot. Each dot represents relative growth or viability of one organoid. (D) Bar graphs show the relative gene expression of AXIN2 by qRT-PCR in primary cell organoids (BCa #04) treated with DMSO or CHIR of 10 µM for 96 hours. GAPDH was used as a reference gene, and relative expression was calculated by the 2 ΔΔCT method. (E) A bar graph shows the relative gene expression of AXIN2 by qRT-PCR in primary cell organoids of 3 pooled patient samples (BCa #02, #03, #04). The result was normalized to the expression of DMSO-treated organoids.^*P ≤ 0.05. ^**P ≤ 0.01. ^***P ≤ 0.001. ^****P ≤ 0.0001. (F) The expression and subcellular localization of β-catenin in primary cell organoids (BCa #04) treated with DMSO or 10 µM CHIR for 72 hours was detected by immunofluorescence staining. Nuclei and actin fibers were counterstained with DAPI and ActinRed, respectively. Scale bar 20 µm.