Figure 4.

Antagonism of p38 inhibitor towards ERK and JNK inhibitor-mediated fibroblast activation. CRL-2097 human dermal fibroblasts were cultured under control conditions or in the presence of 10 µM U0126 (ERKⁱ), 10 µM SP600125 (JNKⁱ), and/or 10 µM SB202190 (p38ⁱ), and analyzed at day 4 in culture. (A) Expression levels of myofibroblast-associated transcripts ACTA2, CNN1 and TAGLN were determined relative to fibroblasts cultured under control conditions by reverse transcription-quantitative polymerase chain reaction. GAPDH expression was used as an internal control. Student’s t-tests were performed in order to compare the expression between each culture condition and its p38ⁱ-treated counterpart. ^*P<0.05, ^***P<0.001 and ^****P<0.0001. There were 4 biological replicates/condition. Error bars depict standard deviation. (B) Protein lysates were examined for expression of myofibroblast-associated marker proteins α-SMA, calponin and SM22α by western blot analysis. Histone H3 was used as a loading control. (C) Fibroblasts were fixed and stained for the myofibroblast marker protein α-SMA and nuclei were counterstained with Hoechst. Scale bars, 100 µm. ACTA2, α-SMA; TAGLN, transgelin; CNN1, calponin 1; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; α-SMA, smooth muscle α actin; ⁱ, inhibitor.