Fig. 5

Fig. 5

Endogenous-TGFβ is necessary and sufficient in driving Tie2-ANGPT signaling.(A) Representative images of ANGPT2 silenced C1-CAF with or without ANGPT1 stimulation (400 ng/ml) for 6 h, detected for pTie2 (Y992) protein. Scrambled siRNA was used as control. (B) qPCR analysis of ANGPT2 following ANGPT2 knockdown in C1 CAF. (C) Quantification of pTie2 (Y992) puncta using ImageJ. (D) Representative images of αSMA and pTie2 (Y992) protein detected by immunofluorescence staining, upon gene silencing of TGFβ, Tie2 and ANGPT1 in TGF-CAF. Scrambled siRNA was used as a control. (E) Myofibroblasts frequency and pTie2 (Y992) puncta was quantified using ImageJ. (F) qPCR analysis of TGFβ, Tie2, ANGPT1 and ANGPT2 followed by knockdown of TGFβ, Tie2 and ANGPT1 in TGF-CAF. (G) Schematic model suggesting experimental design of conditioned media (CM) collection from TGF-CAF following TGFβ, Tie2 and ANGPT1 gene knockdown. (H) Representative images showing myofibroblasts frequency in uninduced C1-CAF exposed to the CM collected from TGF-CAF after TGFβ, Tie2 or ANGPT1 gene-silencing. C1-CAF exposed to C1-CAF CM was used as control. Myofibroblasts frequency was quantified using ImageJ. Scale bar = 20 μm. *P < 0.05, **P < 0.01, ***P < 0.001