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. 2016 Jul 19;11(7):e0159490. doi: 10.1371/journal.pone.0159490

Fig 4. The CXCL12/CXCR4 and TGFβ/TGFβRI axes function independently to promote myofibroblast phenoconversion.

Fig 4

N1 fibroblasts were transfected with 20nM Scramble, anti-CXCR4 (A), and anti-TGFβRI (B) siRNAs for 24 hours and treated with CXCL12 (100pM) for 1 hour. CXCR4 partial knockdown (A) reduced the CXCL12-mediated activation of EGFR, Akt and Erk1/2. However, TGFβRI knockdown (B) had no effect in the CXCL12-mediated activation of EGFR, Akt and ERK1/2. siRNA-mediated knockdown were validated using antibodies against target receptor. Total antibodies for each kinase and actin were used as loading control (Western blot quantification is provided in S1 Fig). N1 fibroblasts were transfected with 20nM Scramble, anti-CXCR4, and anti-TGFβRI siRNAs for 24 hours and treated with CXCL12 (100 pM) (C) and TGFβ (4 ng/mL) (D) for 24 hours. qRT-PCR analysis of α-smooth muscle actin (ACTA2), collagen 1α1 (COL1α1) and TGFβ (TGFβ 1) was performed. (E) Western blot analysis to confirm effective knockdown of CXCR4 and TGFβRI during gene expression analysis. * = p-Value <0.05, ** = p-Value < 0.001. Error bars, SE.