Fig 1: The luciferase assay shows that fortilin blocks TGF-ß1-induced activation of the Smad2/3 binding element.strep-tag-fortilin, recombinant fortilin with strep-tag at its N-terminus; strep-tag-luciferase, recombinant luciferase with strep-tag at its N-terminus, used as the control; a-TGF-ß1 mAb, neutralizing anti-TGF-ß1 monoclonal antibody (1.25 µg/mL or 8.33 nM); HEK, human embryonic kidney cells; SBE-Luc, a vector containing the Smad2/3 binding element fused to the luciferase cDNA; A.U., arbitrary unit; SBE-SEAP, a vector containing the Smad2/3 binding element fused to the secreted embryonic alkaline phosphatase cDNA; MFB-F11SBE-SEAP cells, immortalized mouse embryonic fibroblasts from Tgfb1-/- mice that stably harbor the SBE-SEAP construct; data points, means ± SD; statistical analyses performed using ANOVA with Fisher’s multiple comparison; N, the number of biological replicates; NS, not statistically significant; ****P < 0.001. a Fortilin prevented TGF-ß1 from activating the SBE in HEK293SBE-Luc cells; 1 nM TGF-ß1 and 1 nM strep-tag-fortilin were used. N = 4. b Fortilin, but not luciferase control protein, dose-dependently blocked TGF-ß1-induced SBE activation in the MFB-F11SBE-SEAP cells; 156 pM TGF-ß1 was used to stimulate the cells. Moreover, 19.5 (low dose, +) and 195 (high dose, ++) nM strep-tag-fortilin or strep-tag-luciferase were used to block TGF-ß1-induced SBE activation. N = 6.
Fig 2: Comparison of transcriptome and chromatin accessibility between 786-O cells with and those without DPF3 expression.a The MA plot comparing gene expression profile of 786-O cells with and those without DPF3a expression. The x-axis and y-axis represent Log2 (mean read counts) and Log2 (fold change), respectively. Red dots indicate upregulated genes, whereas blue dots indicate downregulated genes. The results represent two independent biological replicates. b The “Diseases & Functions” analysis of genes upregulated after DPF3a overexpression using Ingenuity Pathway Analysis (IPA) software (QIAGEN). The p value was calculated using the right-tailed Fisher’s exact test. The results represent two independent biological replicates. c The protein level of EMT-related genes SNAI1, ZEB1, and MMP2 in DPF3-OE and control 786-O cells was analyzed by immunoblotting (IB). Three independent experiments were performed and similar results were obtained. d Effect of DPF3a overexpression on MMP2 activity in 786-O cells. MMP2 activity was determined using a fluorogenic substrate as described in Methods. Data were presented as mean values ± SD (n = 3 independent experiments). Statistical significance was estimated using a two-sided student t-test. e The “Upstream Regulator” analysis of genes upregulated after DPF3a overexpression using IPA. The p value was calculated using the right-tailed Fisher’s exact test. The x-axis represents the number of DPF3a regulated genes which were predicted to be downstream targets of specific regulators. The y-axis represents −Log10 p value of overlap between DPF3a regulated genes and downstream targets genes of a specific regulator. The results represent two independent biological replicates. f The migration and invasion ability of 786-O cells after TGFB1 treatment (20 ng/ml) was measured using Transwell assay. Three independent experiments were performed and similar results were obtained. g Scatter plot comparing ATAC-seq normalized read counts (CPM counts per million) at the promoters of genes differentially expressed in 786-O cells versus those without DPF3a overexpression. Pearson’s correlation test was performed. The blue and red dash line represents the fold change of 1.5 and 2, respectively. The results represent one biological replicate. Source data are provided in the Source Data file.
Fig 3: Quantitative western blots and ELISA show that fortilin blocks TGF-ß1-induced phosphorylation of Smad3.SBE-SEAP, a vector containing the Smad2/3 binding element fused to the secreted embryonic alkaline phosphatase cDNA; MFB-F11SBE-SEAP cells, immortalized mouse embryonic fibroblasts from Tgfb1-/- mice that stably harbor the SBE-SEAP construct; strep-tag-fortilin, recombinant fortilin with strep-tag at its N-terminus (19.5 nM); strep-tag-luciferase, recombinant luciferase with strep-tag at its N-terminus, used as the control (19.5 nM); a-TGF-ß1 mAb, neutralizing a-TGF-ß1 monoclonal antibody (1.25 µg/mL or 8.33 nM); IB, immunoblot; a-P-Smad3, anti-phosphorylated Smad3 antibody; a-Smad3, anti-Smad3 antibody; TCE, 2,2,2-trichloroethanol; A.U., arbitrary unit. a Western blot analysis using a-P-Smad3 and a-Smad3 and total protein visualization by TCE. b, c Quantification of P-Smad3, normalized to total proteins (b) and total Smad3 (c), showing that fortilin, but not luciferase control protein, prevented TGF-ß1 from phosphorylating Smad3. d ELISA of P-Smad3 on the lysates from MFB-F11SBE-SEAP cells also showed that fortilin, but not luciferase control protein, prevented TGF-ß1 from phosphorylating Smad3.
Fig 4: WKYMVm treatment maintains and enhances PD-MSC characteristics and effects. (A) Morphology of the PD-MSCs and WKYMVm-treated PD-MSCs. Scale bar = 100 μm. (B) Viability of the PD-MSCs and WKYMVm-treated PD-MSCs shown by CCK-8 assays. (C) Expression of stemness-related markers in the WKYMVm-treated PD-MSCs shown by RT-PCR. (D) MSC surface markers (e.g., hematopoietic, nonhematopoietic, and HLA family) in the WKYMVm-treated PD-MSCs using FACS analysis. (E) Lipid droplets in PD-MSCs differentiated into adipocytes shown by Oil Red O staining. Scale bar = 10 μm. Expression of adipogenic-specific markers such as CFD (F) and PPARG (G) shown by qRT-PCR. (H) Calcium deposition of PD-MSCs differentiated into osteocytes shown by von Kossa staining. Scale bar = 40 μm. Expression of osteogenic-specific markers, including BGLAP (I) and COL1A1 (J), shown through qRT-PCR. (K) Expression of FPR2 in PD-MSCs using immunofluorescence. Scale bar = 25 μm. (L) Quantification of the fluorescence intensity of FPR2 through immunofluorescence. Expression of VEGF (M), HGF (N), and TGFB1 (O) in the WKYMVm-treated PD-MSC culture supernatants shown by ELISAs. (P) The activity of MMP9 in the WKYMVm-treated PD-MSC culture supernatant shown using gelatin zymography. Mean ± SD, * p < 0.05 by t tests. si-FPR2, siRNA-FPR2-transfected PD-MSCs; +WK, WKYMVm-treated PD-MSCs.
Supplier Page from Abcam for Human TGF beta 1 ELISA Kit