Fig 2: The serum concentration of TGF-β was elevated and TGF-β signaling was activated in CRF patients (A) Serum concentrations of TGF-β were associated with the CRF stage. Circulating levels of TGF-β were measured in serum samples obtained from healthy controls (n = 24) and CRF patients at 5 different stages (1-5, n=24 in each stage) *P < 0.05 and *** P < 0.001. (B) Serum concentrations of TGF-β were elevated in CRF patients. Circulating levels of TGF-β in CRF patients (n=120) were combined and compared with levels in controls. *** P < 0.001. (C) The relative expression level of TGFB1 was associated with the CRF stage. The qRT-PCR analyses were performed to measure TGFB1 mRNA levels in renal biopsies from renal cell adenocarcinoma patients (TNM stage 1, control) and 120 CRF patients representing 5 different stages (n=24 in each stage). *P < 0.05, **P < 0.01 and *** P < 0.001. (D) The relative expression level of TGFB1 was elevated in CRF patients. The relative expression levels of TGFB1 in CRF patients (n=120) were combined and compared with levels in controls. ** P < 0.01. (E and F) TGF-β signaling was activated in CRF patients. (E) Western blotting was performed to measure pSmad2, Smad2, pSmad3 and Smad3 in renal biopsies from one renal cell adenocarcinoma patient (TNM stage 1, control) and 5 CRF patients (n=1 in each CRF stage). GAPDH was used as a loading control. (F) The relative protein levels in (E) were quantified and normalized to GAPDH. *P < 0.05, **P < 0.01 and *** P < 0.001.

Fig 3: (A) Overall and (B) progression-free survival time of patients stratified according to p-Smad2/Smad4 co-positive and co-negative expression. Smad4, SMAD family member 4; p-Smad2, phosphorylated SMAD family member 2.

Fig 4: a THC upregulated the expression of claudin-4 compared with that in the control group. b, c Quantitative real-time PCR indicated that THC decreased the expression of c-Jun and Smad2 (*p < 0.05, **p < 0.01, ***p < 0.001). n = 3/group

Fig 5: Tpl2−/− primary keratinocytes are refractory to TGFβ mediated growth inhibition despite having normal levels of TGFβ receptors and SMAD proteins.Primary keratinocytes were cultured in low calcium (0.05 mM) growth media or transduced with v-rasHa (RAS). Real-time PCR (a) and Western analyses (b) were conducted for TGFB, TGFBRI and TGFBRII, SMAD2 and SMAD4. c Fibroblast protein was collected from newborn WT and Tpl2−/− mice and used for Western analysis. All expression was normalized to a housekeeping gene. d WT and Tpl2−/− keratinocytes were treated with 10 ng/ml TGFβ and after 24 h, proliferation was determined using a WST-1 assay. e Whole mRNA was isolated and real-time PCR performed and analyzed for relative abundance of Fas mRNA