Fig 2: FSH upregulates TGF-β signaling molecules in MGCs.MGCs collected from prepubertal mice were cultured for 24 h in cell culture medium without or with 10 ng/ml FSH. a Effects of FSH on Tgfb1, Tgfb3, Tgfbr1, Tgfbr2, Smad2, Smad3, and Smad4 mRNA levels in MGCs. b Effects of FSH on the protein levels of TGFB1, TGFBR2, SMAD2/3, SMAD4, and phosphorylated SMAD2/3 (p-SMAD2/3) in MGCs. The blots shown are representative of three images captured. GAPDH was used as a loading control. c Immunofluorescence analysis for TGFB1, TGFBR2, SMAD2/3, SMAD4, and phosphorylated SMAD3 (p-SMAD3) in MGCs. The images are representative of three images captured. Scale bars: 50 μm. Bars indicate the mean ± SEM of three independent replicates. Values not indicated by the same letter are significantly different (P < 0.05)

Fig 3: TRIM47 overexpression promotes 5′-fluorouracil resistance of colorectal cancer cells in vitro and in vivo. a and b Cell proliferation assay was performed in SW480 cells transfected with vector plasmids, TRIM47 overexpression plasmids and SMAD4 overexpression plasmids (n = 3, non-parametric Mann-Whitney test, P < 0.05). c Cell transwell invasion assay was performed in SW480 cells transfected with vector plasmids, TRIM47 overexpression plasmids and SMAD4 overexpression plasmids (n = 3, non-parametric Mann-Whitney test, P < 0.05). d Cell proliferation assay was performed in SW480 cells treated with increasing concentrations of 5-FU. (n = 3, non-parametric Mann-Whitney test, P < 0.05). e Cell proliferation assay was performed in SW480 cells transfected with vector plasmids, TRIM47 overexpression plasmids and 5-FU (n = 3, non-parametric Mann-Whitney test, P < 0.05). f Representative images of tumors in nude mice bearing colorectal cancer cells treated with control adenovirus, TRIM47 overexpression adenovirus, 5-FU, TRIM47 overexpression adenovirus & 5-FU (n = 8). g Tumor volume was measured after different treatments in nude mice (n = 8, non-parametric Mann-Whitney test, P < 0.05). h Tumor weight was measured in nude mice after different treatments (n = 8, non-parametric Mann-Whitney test, P < 0.05). i A schematic model of TRIM47 function in colorectal carcinogenesis. TRIM47 interacts with SMAD4 and promotes the ubiquitination and degradation of SMAD4, impairing the suppression of CCL15 and CCR1, ultimately promoting colorectal cancer cells proliferation and metastasis

Fig 4: Correlation between INMT and tumor signaling pathways. (A,B) In TCGA and GSE46602, INMT was enriched in prostate cancer, MAPK signaling, TGFβ signaling, and Wnt signaling pathways. After transfection with corresponding negative control lentiviral vector and INMT overexpression lentiviral vector in 22Rv1 and PC-3, (C) mRNA levels of INMT were measured using real-time PCR, and (D,E) protein levels of INMT were measured using western blot. (D,F) Protein levels of MAPK signaling pathway-related molecules (Phospho-p38 MAPK, p38 MAPK) were measured using western blotting. (D,G) Protein levels of TGFβ signaling pathway-related molecules (TGFβ1, Smad2, and Smad4) were measured using western blot. (D and H) The protein levels of Wnt/β-catenin signaling pathway-related molecules (β-catenin and cyclin D1) were measured using western blot. The samples derive from the same experiment, and those blots were processed in parallel.

Fig 5: Generation of a double SMAD2/SMAD3 knockout BxPC-3 (SMAD4-negative) pancreatic cancer cell line.a Immunoblot of phospho-SMAD2 (pSMAD2), SMAD2, phospho-SMAD3 (pSMAD3), SMAD3 and SMAD4 after treatment with TGF-β and/or TGF-βR1 kinase activity inhibitor (RepSox) for 1 h on cytoplasmic (GAPDH as control) and nuclear fractions (phospho-histone H3 (pHIS-H3) as control) prepared from SMAD4-positive PANC-1 cells (left panel) and SMAD4-negative BxPC-3 cells (right panel). One representative image out of 3 independent experiments is shown. b Immunoblot of SMAD2, SMAD3, SMAD4 and GAPDH on PANC-1 (SMAD4+), BxPC-3 (SMAD4-) wild-type (WT), control (Ctrl) and SMAD2/SMAD3-knockout (S2/3-KO) cells obtained by CRISPR/Cas9 genome editing technique. One representative image out of 3 independent experiments is shown. c Flow cytometric analysis of SMAD2/3 performed on control and S2/3-KO BxPC-3 cells. SMAD2/3 ratio was determined as the mean value of three independent experiments. d Immunofluorescent micrographs of SMAD2/SMAD3 staining on control and S2/3-KO BxPC-3 cells treated with TGF-β (1 h). Nuclei were counterstained in blue with DAPI. Representative images out of 3 independent experiments are shown. Scale bar (left-hand images) = 50 µm. Scale bar (right-hand images) = 20 µm.