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. 2020 Aug 18;15(8):e0232917. doi: 10.1371/journal.pone.0232917

Fig 4. Sunitinib inhibits TGF-β1-induced expression of EMT markers and Smad signaling.

Fig 4

(A) Serum-deprived A549 cells were treated with TGF-β1 (5 ng/mL) or its combination with sunitinib for 72 h. The protein expression of TGF-β1-mediatead EMT markers was evaluated by Western blot. Actin was used as a loading control. (B and C) Serum-deprived A549 cells were pretreated with sunitinib for 3 h, and then incubated with TGF-β1 (5 ng/mL) for 48 h. After RNA extraction and cDNA synthesis, we performed qRT-PCR to measure mRNA expression of CDH2 and VIM using GAPDH as an internal control. (D) Effects of sunitinib on TGF-β1-activated Smad signaling. A549 cells were treated with TGF-β1 (5 ng/mL) or its combination with sunitinib for 24 h, and then the levels of p-Smad2/3 and endogenous Smad2/3 in cytosolic fraction were evaluated by Western blot. (E and F) The transcriptional activity of SNAI1 and TWIST1 was measured by qRT-PCR using GAPDH as an internal control. Serum-deprived A549 cells were pretreated with sunitinib for 3 h, and then incubated with TGF-β1 (5 ng/mL) for 1 h. (G) The effect of sunitinib on TGF-β1-induced phosphorylation of ERK1/2 and p38 was determined by Western blot. Actin was used as a loading control. # p < 0.01 versus control, * p < 0.05, ** p < 0.01 versus treatment with TGF-β1 only.