Fig 2: Downregulation of SMAD2 and SMAD4 reverse the miR-19a-3p-downregulation-induced invasion and migration of PCa cells. (A) The stimulatory effects of silencing miR-19a-3p on the transcriptional activity of a TGF-ß/Smad-responsive luciferase reporter were abrogated by individual downregulation of SMAD2 and SMAD4. Error bars represent the mean ± SD of three independent experiments. *P<0.05. (B and C) The stimulatory effects of silencing miR-19a-3p on the invasion and migration abilities of PCa cells were abrogated by individual downregulation of SMAD2 and SMAD4. Error bars represent the mean ± SD of three independent experiments. *P<0.05.

Fig 3: Hyperphosphorylation of CaMKII, SMAD2, and MAPK in LRT. A–E: Phosphorylation of proteins involved in calcium, MAPK, and TGF-ß MAPK signaling were measured by Western blotting in lysates from gastrocnemius muscle under resting conditions (basal) or immediately after an acute bout of exercise (exercise). ERK, extracellular signal–regulated kinase; P38, p38 mitogen-activated protein kinase; SMAD, mothers against decapentaplegic homolog. n = 6–7/group. *P < 0.05 for phenotype main effect; ^P < 0.05 for exercise main effect; °P < 0.05 for phenotype–exercise interaction by 2-way ANOVA. P values obtained by Tukey post hoc testing are displayed. AU, arbitrary units.

Fig 4: miR-505-3p targets SMAD2 and SMAD3 in PCa cells. (A) Predicted miR-505-3p targeting sequence and mutant sequences in the 3'UTRs of SMAD2 and SMAD3. (B) Real-time PCR analysis of SMAD2, SMAD3 and TGFBR1 expression in the indicated cells. *P<0.05. (C) Western blot analysis of SMAD2 and SMAD3 expression in the indicated cells. a-tubulin served as the loading control. (D-F) Luciferase assay of the 3'UTR reporter of SMAD2 and SMAD3 in the indicated cells. *P<0.05. (G-I) MiRNP IP assay revealing the association between miR-505-3p and SMAD2 and SMAD3 transcripts in PCa cells cells. Pulldown of the IgG antibody served as the negative control. *P<0.05. PCa, prostate cancer; 3'UTRs, 3'untranslated regions.

Fig 5: SMAD2 and SMAD3 are crucial in the migratory and invasive potential of BxPC-3 (SMAD4-negative) cells in response to TGF-ß.a Images of a transwell migration assay with control (Ctrl), S2-KO, S3-KO, and S2/3-KO genetically engineered BxPC-3 cells cultured for 24 h in the presence or absence of TGF-ß. Scale bar = 3 mm. Graphs represent mean values +/– SEM (n = 6). One-tailed Wilcoxon/Mann–Whitney test; **p-value < 0.01; *p-value < 0.05; ns, not significant. Scale bar = 3 mm. b Images of a transwell invasion assay with control (Ctrl), S2-KO, S3-KO, and S2/3-KO genetically engineered BxPC-3 cells cultured for 72 h in the presence or absence of TGF-ß. Scale bar = 3 mm. Invasion was quantified as the mean value of six independent experiments and represented as a graph. Mean values +/– SEM are represented. One-tailed Wilcoxon/Mann–Whitney test; **p-value < 0.01; *p-value < 0.05; ns, not significant. c Images (left panel) of zebrafish embryos (fli:GFP) grafted with pre-stained control or S2/3-KO BxPC-3 cells (in red). Zebrafish embryos were incubated at 28 °C for 24 h after engraftment, and then imaged on an EVOS Cell Imaging System microscope. White arrows point toward disseminated cells (in red). Scale bar (left-hand images) = 1 mm. Scale bar (right-hand images) = 0.25 mm. Right panel: quantification of invaded metastatic cells per embryo, performed 24 h post-engraftment. Mean values (horizontal bars) +/– SD are represented. One-tailed Wilcoxon/Mann–Whitney test; ***p-value < 0.001. d Immunoblot of phospho-SMAD2 (pSMAD2), SMAD2, phospho-SMAD3 (pSMAD3), SMAD3 and GAPDH on control (BxPC-3 Ctrl), S2/3-knockout (BxPC-3 S2/3-KO) and S2/3-Rescue (BxPC-3 S2/3-Rescue) cells treated or not with TGF-ß for 1 h. One representative image out of 3 independent experiments is shown. pSMAD/SMAD ratios were quantified from 3 independent experiments and represented as graphs of mean values +/– SD. e Images of a transwell migration assay with control, S2/3-KO, and S2/3-Rescue BxPC-3 cells cultured for 24 h in the presence or absence of TGF-ß. Scale bar = 3 mm. Graphs represent mean values +/– SEM of five independent experiments. One-tailed Wilcoxon/Mann–Whitney test; **p-value < 0.01; *p-value < 0,05; ns, not significant. f Images of a transwell invasion assay with control, S2/3-KO, and S2/3-Rescue BxPC-3 cells cultured for 72 h in the presence or absence of TGF-ß. Scale bar = 3 mm. Graphs represent mean values +/– SEM of five independent experiments. One-tailed Wilcoxon/Mann–Whitney test; **p-value < 0.01; *p-value < 0,05; ns, not significant.