Fig 2: P311 deficiency down-regulates TGF-β/Smad signaling in UUO mice.(A) TβR I mRNA expression was determined by real-time PCR in kidneys from P311+/+ (n = 6) and P311−/− (n = 6) after mice 7 days after UUO or sham operation. (B) TβR II mRNA expression was determined by real-time PCR in kidneys from P311+/+ (n = 6) and P311−/− (n = 6) mice 7 days after UUO or sham operation. (C) p-Smad2, p-Smad3, Smad2, Smad3, Smad4, Smad7 and GAPDH protein levels were determined in kidneys by western blot. (D) Relative density of Smad2 protein (n = 3 per group) in each treatment group. (E) Relative density of Smad3 protein (n = 3 per group) in each treatment group. (F) Relative density of p-Smad2 protein (n = 3 per group) in each treatment group. (G) Relative density of p-Smad3 protein (n = 3 per group) in each treatment group. (H) Relative density of Smad4 protein (n = 3 per group) in each treatment group. (I) Relative density of Smad7 protein (n = 3 per group) in each treatment group. A-I are representative of at least three similar experiments. Data are presented as the mean ± SD. *P < 0.05; **P < 0.01.

Fig 3: The supernatant of mesenchymal fibroblasts promotes collagen expression in keloid fibroblasts.a Isolation of CD266+CD9− and other fibroblasts from keloid dermis by flow cytometry. b qRT-PCR assay of some mesenchymal fibroblast marker genes expression in CD266+/CD9− fibroblasts and other fibroblasts from keloid dermis. Data are presented as mean values ± SD (n = 3 biologically independent experiments.). Two-sided unpaired t-test, ***P < 0.001(ASPN:P = 0.00003, COL11A1:P = 0.00019, POSTN:P = 0.00004, ADAM12:P = 0.00015, P311:P = 0.00019, COMP:P = 0.00002). c Western blot assay of mesenchymal fibroblast marker genes expression in CD266+/CD9− fibroblasts and other fibroblasts from keloid dermis. The experiments were repeated three times with three different fibroblast donors, and here a representative result was shown. d GO Biological Process enrichment analysis of differentially expressed genes between keloid CD266+/CD9− fibroblasts and other fibroblasts. e GSEA enrichment plots for representative signaling pathways upregulated in keloid CD266+/CD9− fibroblasts compared to other fibroblasts. (NES normalized enrichment score, corrected for multiple comparisons using FDR method, P-value were showed in plots). f, g Keloid other fibroblasts were treated with the supernatant of CD266+/CD9− or other fibroblasts. The expression of collagen I and collagen III was analyzed by qRT-PCR (f) or western blot (g). Data are presented as mean values ± SD (n = 3 biologically independent experiments). Two-sided unpaired t-test, **P < 0.01, ***P < 0.001. (COL1A1: P = 0.000009 and P = 0.000015, respectively; COL3A1: P = 0.00006 and P = 0.00214, respectively). The experiments were repeated three times with three different fibroblast donors, and here a representative result was shown. h Keloid other fibroblasts were treated as indicated in the figure. The expression of collagen I and collagen III was analyzed by western blot. The POSTN antibody neutralizing experiments were repeated three times with three different fibroblast donors, and here a representative result was shown.

Fig 4: P311-induced EpMyT is mediated by TGFβ1/Smad signaling. a Western blot analysis showing expression of LAP-TGFβ1 and active TGFβ1 in primary mouse EpSCs. Relative densities of target proteins are also shown in comparison with the density of GAPDH. **P < 0.01. b Real-time PCR showing TGFβ1 mRNA levels in P311-transfected and control vector-transfected EpSCs. *P < 0.05. c Real-time PCR showing TβRI and TβRII mRNA levels in P311-transfected and control vector-transfected EpSCs. *P < 0.05 and **P < 0.01. d Phase-contrast microscopic observations of cell morphology 72 hours after the EpSCs were transfected with P311-containing vectors either with or without 5 μM LY2109761. e Immunofluorescence was performed to determine the expression of α-SMA (upper) and E-cadherin (lower) in EpSCs at 72 hours after transfection with P311 (left), the control vector (middle), and P311 with LY2109761 (right). α-SMA and E-cadherin were labeled using AF594 (red), and nuclei were stained using DAPI (blue). Scale bar, 25 μm. f Quantification of α-SMA+ and E-cadherin+ cells in an average of five regions. **P < 0.01 for P311 vs control vector; ##P < 0.01 for P311 vs P311 + LY2109761. g Western blot analysis was performed to determine α-SMA, vimentin, β1-integrin, and E-cadherin levels. *P < 0.05 and **P < 0.01 for P311 vs control vector; and #P < 0.05 and ##P < 0.01 for P311 vs P311 + LY2109761. h Western blot analysis showing total and phosphorylated levels of Smad2 and Smad3 in primary EpSCs. LY2109761 was observed to have an inhibitory effect on Smad signals. *P < 0.05 and **P < 0.01 for P311 vs control vector; and ##P < 0.01 for P311 vs P311 + LY2109761. i Western blot analysis was performed to determine α-SMA, E-cadherin, and total and phosphorylated Smad3 levels after stimulation of Smad3 siRNA.*P < 0.05 and **P < 0.01 for P311 or P311 + mock siRNA vs the control vector; and ##P < 0.01 for P311 + Smad3 siRNA vs P311 or P311 + mock siRNA. j ELISA showing the level of total TGFβ1 in the culture supernatants of P311-transfected and control vector-transfected EpSCs. *P < 0.05. k Western blot analysis showing the expression of α-SMA in primary P311 WT and P311 KO EpSCs after the cells were incubated with or without TGFβ1 treatment. *P < 0.05 and **P < 0.01 relative to PBS control; ##P < 0.01 relative to P311 KO cells treated with TGFβ1. l Western blot analysis was performed to determine the total and phosphorylated Smad3 levels in P311 KO and P311 WT EpSCs with or without exogenous TGFβ1. Data shown as the mean ± SEM. **P < 0.01 relative to PBS control; #P < 0.05 relative to P311 KO cells treated with TGFβ1 (Color figure online)

Fig 5: P311 might be involved in macrophage infiltration in UUO mice.(A) Representative F480-stained sections of UUO kidneys from P311+/+ (n = 6) and P311−/− (n = 6) mice. Black arrowhead indicates the F480-positive cells. Bottom panel: negative controls for the immunohistochemical staining of F480 on the obstructed kidneys from both P311+/+ and P311−/− mice. (B) The graph represents the total number of F480-positive sections from five contiguous high-powered fields per kidney section. (A) and (B) are representative of at least three similar experiments. Scale bar: 100 μm. Data are presented as the mean ± SD. *P < 0.05; **P < 0.01.