Fig. 2
ALDH1A1 and ALDH1A3 regulate TGFB1 gene expression through RA-dependent mechanism. A Relative mRNA expression of TGFB1 gene in LNCaP, C4-2B and PC3 cells upon knockdown of ALDH1A1 or ALDH1A3 expression. Cells transfected with scrambled siRNA (siScr) were used as a control. n ≥ 3; Error bars = SD; *p < 0.05; ***p < 0.001. B Distribution of log2 fold change values (relative to siScr control) for TGFB1 up or down genes. The gene sets for the TGFB1 responsive genes were described previously [59]. Values are sorted from high to low in each column independently; therefore, the order of gene names is not the same. Significance of enrichment for up- or down-regulated genes evaluated by Wilcoxon signed rank test; *p < 0.05; **p < 0.01; ***p < 0.001; n.s. – non-significant. C Correlation of ALDH1A1 and ALDH1A3 expression with TGFB1 mRNA levels in the PCa gene expression datasets TCGA PRAD (n = 493), MSKCC primary (n = 131), Broad/Cornell (n = 31), DKFZ (n = 118), and SMMU (n = 65) and non-cancerous tissues (MSKCC cohort, n = 29); *p < 0.05; n.s.- non significant. D Correlation of RXRA and RARA expression with TGFB1 mRNA levels in the PCa gene expression datasets TCGA PRAD (n = 493), MSKCC primary (n = 131), Broad/Cornell (n = 31), DKFZ (n = 118), and SMMU (n = 65) and non-cancerous tissues (MSKCC cohort, n = 29); *p < 0.05; n.s.- non significant. E LNCaP, C4-2B or PC3 cells were treated with various concentrations of ATRA (10 μM, 25 μM, and 50 μM) or DMSO as a control for 48 h. Relative mRNA expression of ALDH1A1, ALDH1A3 and TGFB1 was measured by qPCR analysis. n ≥ 3; Error bars = SD; *p < 0.05; **p < 0.01; ***p < 0.001. F 22Rv1 cells were transfected with pGL3-TGFB1 luciferase reporter plasmid [44] and treated with various concentrations of ATRA or 9CisRA (10 μM, 25 μM, and 50 μM) and DMSO as a control. Luciferase activity was measured 48 h after the treatment started. n ≥ 3; Error bars = SD; *p < 0.05. G qPCR analysis of TGFB1 expression in LNCaP cells upon transient RARA overexpression, treatment with ATRA or 9CisRA for 48 h or both. Cells transfected with empty plasmid were used as control. n ≥ 4; Error bars = SD; **p < 0.01; ***p < 0.001; ****p < 0.0001; n.s.—non significant