Fig 2: RGMa enhances Slug through MEK1/2-ERK1/2 signaling pathway. A: Western blot analysis for the expression of p-ERK1/2, ERK1/2, p-MEK1/2, and MEK1/2 in the primary rat VSMCs treated with 10 µg/ml ox-LDL for 24 h. n = 6. B, C: Western blot analysis (B) and quantification (C) for the expression of RGMa, Slug, CD68, a-SMA, and SM22a in the VSMCs treated with 10 µg/ml ox-LDL for 24 h. n = 6; **P < 0.01. D–I: Western blot analysis (D) and quantification for the expression of Slug (E), RGMa (F), CD68 (G), a-SMA (H), and SM22a (I) in the VSMCs treated with PD98059 for 2 h and/or followed by 10 µg/ml ox-LDL for 24 h. n = 4; *P < 0.05, **P < 0.01; ns indicates not significant. J–O: Western blot analysis (J) and quantification for the expression of Slug (K), RGMa (L), CD68 (M), a-SMA (N), and SM22a (O) in primary rat VSMCs treated with different conditions (ox-LDL + pcDNA-NC, ox-LDL + pcDNA-RGMa, ox-LDL + pcDNA-NC + PD98059, and ox-LDL + pcDNA-RGMa + PD98059). n = 4; *P < 0.05, **P < 0.01, ns indicates not significant.

Fig 3: SMYD3 knock down or pharmacological blockade tempers MDA-MB-231 mesenchymal phenotype and migration ability. (A) SMYD3 was knocked down by siRNA transfection in MDA-MB-231 cells, total extracts were collected after 48 h and assayed for epithelial (E-cad) or mesenchymal (Fibronectin, Vimentin and Snail1) protein levels by immunoblot. GAPDH was used as a loading control. nFold is relative to DMSO treated cells. (B) MDA-MB-231 cells were treated with increasing doses of BCI121 (0, 10 or 50 µM) for 48 h. Epithelial (Occludin, Claudin6) or mesenchymal (Snail2, N-cadherin and Fibronectin) marker mRNA levels were measured by qRT-PCR. GAPDH was used as housekeeping gene. Data represent means±SD. Statistical analysis was performed with 1 way ANOVA, followed by post-hoc Tukey test n\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$ \ge$\end{document}3, * P = 0.05, ** P = 0.01, *** P = 0.001. (C) MDA-MB-231 cells were treated with increasing doses of BCI121 (0, 10 or 50 µM) for 72 h and whole cell extracts were assayed for mesenchymal (Snail1, Snail2, Vimentin and Fibronectin) protein levels by immunoblot. GAPDH was used as a loading control. (D) Wound-healing assay on MDA-MB-231 cells treated with 10 µM BCI121 for 0 and 16 h. Migration percentage is reported in the right panel. Statistical significance was calculated with Student's t-test. *** P = 0.001. Scale bar = 10µm. (E) in vivo migration assay in zebrafish xenografts. 1 × 102 MDA-MB-231 cells were injected in the subepidermal pocket of zebrafish embryos at 48 hpf. Embryos were treated with 50 µM BCI-121 or DMSO, for 48 h, n = 14 DMSO embryos, n = 12 BCI121 embryos. (F) Cells migrating in the tail, heart and head were counted in the two groups, 48 hpi. Scale bar = 200µm. Statistical analysis was performed with unpaired Student's t-test. * P = 0.05. Normalized band intensity in immunoblots is reported above signals.

Fig 4: The interaction between GSE1 and TACSTD2 drives metastatic disease, castration resistance, and disease progression in patients with prostate cancer. Box-and-dot plots showing the differential expression of (A) GSE1 and (B) TACSTD2 genes in normal, tumor, and metastatic samples from the TCGA PRAD cohort. Box-and-whisker plots showing the correlation between (C) GSE1 or (D) TACSTD2 gene expression and clinical M stage in the TCGA PRAD dataset. (E) Graphical representation of the correlation between GSE1 and TACSTD2 gene expression levels. (F) Principal component analysis of the expression data for primary localized PCa vs. castration-resistant bone metastatic prostate cancer in the GSE32265 cohort. Unit-variance scaling was applied to rows; SVD with imputation was used to calculate principal components. X- and Y-axes show Principal Components 1 and 2, which explain 26.6 and 13.2% of the total variance, respectively. Prediction ellipses were computed such that, with a probability of 0.95, a new observation from the same group would fall inside the ellipse. N = 55 data points. (G) Expression heatmap showing the relationship between metastasis, the TMPRSS2-ERG fusion gene, and expression of GSE1, TACSTD2, KLK3/PSA, and biomarkers of angiogenesis, metastasis, and proliferation in the GSE32265 cohort. Columns with similar annotations were collapsed by taking the mean in each group. Rows are centered; unit-variance scaling was applied to rows. Both rows and columns were clustered using correlation distance and average linkage, with 25 rows and 5 columns. (H) Representative Western blot images of the differential expression of GSE1, TACSTD2, VIM, SNAI2, and VEGFA proteins in HPrEC, LNCaP, PC3, or DU145 cells. GAPDH served as a loading control. FC, fold change; meta, metastatic; norm, normal; M, distant metastasis; M0, no distant metastasis; M1a, distant metastasis to non-regional lymph node(s); M1b, distant metastasis to bone(s); M1c, distant metastasis to other site(s) with or without bone disease; BCR, biochemical recurrence; SVD, singular value decomposition. ** p < 0.01; *** p < 0.001; ns, not significant.

Fig 5: aPD-L1 reverses TNBC malignant evolution in the co-culture of TNBC cells and TAMs. (A and B) MDA-MB-231 and Hs578T cell migration capacity was assessed using wound healing and Transwell assays when the cells were treated with or without aPD-L1 in a co-culture system (magnification, ×200). (C) Protein expression levels of VEGF, MMP2 and MMP9 in MDA-MB-231 and Hs578T cells, with or without aPD-L1 in a co-culture system, were determined using western blot analysis. (D) Protein expression levels of the EMT markers, E-cadherin, ZO-1, N-cadherin, vimentin, Slug and Twist, in MDA-MB-231 and Hs578T cells, with or without aPD-L1 in a co-culture system, were determined using western blot analysis. (E) Protein expression levels of the stemness markers, CD44, Oct4, Nanog, Bmi1 and Sox2 in MDA-MB-231 and Hs578T cells, with or without aPD-L1 in a co-culture system were determined using western blot analysis. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. aPD-L1, programmed death-ligand 1 inhibitor; TNBC, triple-negative breast cancer; TAM/M2, tumor-associated macrophages/M2-type; VEGF, vascular endothelial growth factor; EMT, epithelial-mesenchymal transition; ZO-1, zonula occludens-1.