Fig 2: M1-Mf suppress EMT, migration, and invasion of trophoblasts in vitro. (A) Schematic illustration of the M1-Mf and trophoblast co-culture model and the experimental design. (B) Western blot analysis of E-cadherin (E-cad), N-cadherin (N-cad), and vimentin (Vim) protein levels in HTR-8 and JEG3 cells co-cultured with M1-Mf or control. (C-D) RT-PCR assays of E-cad, N-cad, and Vim mRNAs in HTR-8 and JEG3 cells co-cultured with M1-Mf or control. (E-H) Migration and invasion capacities of trophoblasts (HTR-8 and JEG3) alone or co-cultured with M1-Mf determined by wound healing and transwell assays, respectively. Representative images of migrated or invaded cells are shown (magnification, × 200). Error bars, SD. *P < 0.05, **P < 0.01.

Fig 3: Direct tumor-fibroblast contact triggers activin A secretion leading to EMT of tumor cells and myofibroblast activation.a Human cytokine array used conditional medium from transwell (indirect, ID) versus direct (D) co-culture of BxPC-3 with pancreatic stellate cells (PSCs). Cytokine levels in direct co-cultured condition were normalized to indirect co-cultured condition. b Quantitative real-time PCR analyses of CLDN1, VIM, Snail, Twist1, ZEB1, and ZEB2 expression were performed in BxPC-3 cells treated with recombinant human Activin A (50 and 100 ng/ml) compared to untreated cells. Values were presented as mean ± SD (n = 3). c Western blotting analysis of proteins harvested from BxPC-3 cells treated with recombinant human activin A (50 and 100 ng/ml) compared to untreated cells. d Sphere-formation assay of BxPC-3 cells treated with recombinant human activin A compared to untreated cells. Values were presented as mean ± SD (n = 4). e Western blotting analysis of proteins harvested from mono-cultured and direct co-cultured (D) tumor cells (BxPC-3-RFP) treated with IgG (1 µg/ml) or anti-activin A-neutralizing antibody (1 µg/ml, 693604, Biolegend). FACS was performed to collect RFP+ tumor cells after 48 h of mono- or co-culture conditions. f Sphere-formation assay of BxPC-3-RFP cells in mono-cultured and direct co-cultured conditions treated with IgG (2 µg/ml) or anti-activin A-neutralizing antibody (1 and 2 µg/ml). Values were presented as mean ± SD (n = 4). g, h 3D spheroid invasion assay of BxPC-3-RFP spheroids in mono-cultured and direct co-cultured conditions treated with IgG (4 µg/ml) or anti-activin A-neutralizing antibody (2 and 4 µg/ml). g Representative images. Scale bar, 200 µm. h Extensive rate of each tumor spheroid was calculated by normalizing RFP+ area at 72 h to RFP+ area at 1 h (left panel) and by normalizing RFP+ maximal diameter at 72 h to RFP+ maximal diameter at 1 h (right panel). Values were presented as mean ± SD (n = 9). i, j Subcutaneous injection of BxPC-3 cells and mixed fibroblasts/BxPC-3 cells treated with IgG (1 µg/ml) or anti-activin A-neutralizing antibody (1 µg/ml) every 3 days and collected tumor samples at day 14 after inoculation. Representative images i and quantification results of tumor weight j. Scale bar, 1 cm. Values were presented as mean ± SD (n = 6). k, l Subcutaneous injection of BxPC-3 cells and mixed fibroblasts/BxPC-3 cells treated with recombinant human follistatin 20 ng/ml every 3 days and collected tumor samples at day 14 after inoculation. Representative images k and quantification results of tumor weight l. Scale bar, 1 cm. Values were presented as mean ± SD (n = 5). m, n Quantitative real-time PCR analyses and western blotting analysis of ACTA2 and COL1A1 expression were performed in PSCs treated with recombinant human activin A (50 and 100 ng/ml) compared to untreated cells for 24 h and 48 h, respectively. For qPCR analyses, values were presented as mean ± SD (n = 3). o, p Quantitative real-time PCR analyses and western blotting analysis of ACTA2 and COL1A1 expression in mono-cultured, indirect co-cultured (ID), and direct co-cultured (D) fibroblasts (PSC-GFP) with tumor cells (BxPC-3). FACS was performed to collect GFP+ fibroblasts after 24 h and 48 h for mRNA and protein samples, respectively. For qPCR analyses, values were presented as mean ± SD (n = 3). q Western blotting analysis of proteins harvested from mono-cultured and direct co-cultured (D) fibroblasts (PSC-GFP) treated with IgG (2 µg/ml) or anti-activin A-neutralizing antibody (1 and 2 µg/ml). FACS was performed to collect GFP+ fibroblasts after 48 h of mono- or co-cultured conditions. r, s BxPC-3 cells and mixed PSCs/BxPC-3 cells treated with IgG (2 µg/ml) or anti-activin A-neutralizing antibody (2 µg/ml) every 3 days and collected tumor samples at day 14 after inoculation. Representative IHC images r and H Score s of aSMA expression. Scale bar, 0.5 mm. Values were presented as mean ± SD (n = 6). t, u BxPC-3 cells and mixed PSCs/BxPC-3 cells treated with follistatin (20 ng/ml) every 3 days and collected tumor samples at day 14 after inoculation. Representative IHC images t and H score u of aSMA expression. Scale bar, 0.5 mm. Values were presented as mean ± SD (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (two-tailed t-test).

Fig 4: NR4A1 may be the downstream signal factor of JMJD1A regulating HK-2 cell fibrosis. (A) The expression of transcriptome predictors (PIK3R3, NR4A1, TNC, SEMA7A, CDH1, and F3) after JMJD1A knockout in HK-2 cells by RT-qPCR(n = 3, *p < 0.05). (B,C) RT-qPCR and Western blot were used to detect the mRNA and protein expression of NR4A1 in shJMJD1A and JMJD1A group under AGEs stimulation, compared with shCtrl or vector group, NR4A1 was upregulated in shJMJD1A group, but down regulated in JMJD1A group. (D,E) NR4A1 was inhibited by the inhibitor DIM-C-pPhCO2Me (20 µM), Western blot detected the related fibrosis, and the expression levels of VIM, TGF-ß1, CTGF, and COL1, which were higher in AGEs + DIM group compared with AGEs group (n = 3, *p < 0.05, **p < 0.001).

Fig 5: (A–D) VIM, CDH1, S100A4, EPCAM mRNA expression in GC tissues of SYSU Cohort patient of Distant metastases.