Fig. 2: RBM3 attenuates stem-like properties of PCa cells co-cultured with osteoblasts by inhibiting Wnt/β-catenin activation.

a Left column, representative image of prostate cancer cell spheroidization in MEM-a medium; right column, representative image of prostate cancer cell spheroidization in osteoblast-conditioned medium; Among them, PC-3 was cultured under serum-free condition, and DU145 was cultured under 1% FBS condition; scale bar, 50 µm; Data are mean ± SD (n = 3); *P < 0.05, **P < 0.01. P values were calculated using two-sided paired Student’s t test. b The protein expression of β-catenin, RBM3, and RBM3-Flag were detected by Western Blot in three prostate cancer cells with or without osteoblast co-culture, and the actin levels were used as the internal controls. Semi-quantitative analysis of the bands was shown in Supplementary 1a. c Immunofluorescence staining of the protein expression and subcellular localization of β-catenin and RBM3 of three prostate cancer cells with or without osteoblast co-culture. And the most representative parts were cut out for display. See Supplementary Material for original image (Supplementary 1c, d, e); cells were counterstained with 4’, 6-diamidino-2-phenylindole (DAPI) to reveal nuclei. d Dual luciferase reporter assay of the Wnt signaling activity in three prostate cancer cells. Data are mean ± SD (n = 3); *P < 0.05, **P < 0.01, ***P < 0.001; P values were calculated using two-sided paired Student’s t test; Renilla fluorescence values were used as the internal controls. e RT-qPCR analysis of the mRNA expression of β-catenin and its downstream target genes in PC-3 cells with or without osteoblast co-culture. Data are mean ± SD (n = 3); *P < 0.05, **P < 0.01. P values were calculated using two-sided paired Student’s t test.