Fig 2: SOX4 mediates activation of the PI3K pathway through TGFBR2.a Western blot analysis showing downregulation of the PI3K pathway activity as indicated by decreased expression of PI3K pathway downstream components in HCC1143 following siTGFBR2 treatment (96 h) relative to siControl treatment. p-SMAD2 downregulation is used as a positive control for TGFBR2 protein depletion. b Similar results were observed in HCC1954 cells. c Lentiviral overexpression of TGFBR2 restores pAkt levels in siSOX4 (96 h) treated HCC1143 cells. d Consistent results were observed in HCC1954 cells.

Fig 3: BMP4 induces activated Smad8 in C2C12 myoblasts and suppresses myomiRs. (A) Smad8 mRNA levels were quantitated by qPCR in C2C12 cells after stimulation with BMP4 for different durations in growth media (GM). Smad8 mRNA induction peaked at 24 hours. (B) qPCR results show a dose-dependent repressive effect on miR-1, miR-133a, and miR-133b at 24 h. (C,D) Western blot of C2C12 cells stimulated with BMP4 (300 ng/mL for 24 h) shows induction of P-Smad1/5/8 with a greater than 4-fold increase in P/T-Smad1/5/8 ratio by densitometry. (E,F) Western blot and densitometry of total Smad2/3 (T-Smad2/3) and phosphorylated Smad2 (P-Smad2) show no significant induction in BMP4-stimulated cells. Bars show mean ± SEM of 3 biological replicates per group. p values (compared to vehicle): % < 0.05, # < 0.01, & < 0.001, **** < 0.0001, and θ < 0.0001. RQ, relative quantity. RU, relative units.

Fig 4: Effects of TA on constitutive and TGF-β2-stimulated signaling activities in cultured human RPE cells. (A) Human R-50 cells were treated with TA at 10 µM under serum-supplemented or -starved conditions for the indicated periods. Total proteins were subjected to western blot analysis of p-Smad2, p-ERK1/2 and p-p38 MAPK. (B) Densitometric analysis of (A). (C) R-50 RPE cells received 2 h of serum starvation, followed by treatment with 10 ng/ml TGF-β2 in the presence or absence of 10 µM TA. (D) Densitometric analysis of (C). Data are presented as the mean ± SD from three independent experiments. #P<0.05 vs. respective 0 h group; *P<0.05 vs. respective TA (−) group. RPE, retinal pigment epithelial; TA, triamcinolone acetonide; TGF-β2, transforming growth factor-β2; p, phosphorylated; ERK1/2, extracellular-regulated kinase 1/2; MAPK, mitogen-activated protein kinase.

Fig 5: The Nodal signaling pathway.Genetic interaction and co-Immunoprecipitation (co-IP) studies have shown that the TGFß family ligand Nodal (orange) interacts with the extracellular domains of type I Activin receptor-like kinases, including ALK4 and/or ALK7 (light blue), and type II Activin receptor kinases, including ACTRIIA and/or ACTRIIB (dark blue). Simultaneous binding of Nodal to both receptors initiates a phosphorylation cascade that leads to translocation of phosphorylated Smad2 or Smad3 (light green) transcription factors to the nucleus and expression of Nodal target genes. Nodal signaling also requires the EGF-CFC ‘co-receptors’ Cripto-1 and/or Cryptic (purple) during embryonic development. Co-IP studies have shown that EGF-CFC co-receptors interact with Nodal, but how they mediate Nodal signaling is not well understood. The secreted protein Cerberus (light orange) binds Nodal and inhibits Nodal signaling.