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. 2018 Feb;58(2):216–231. doi: 10.1165/rcmb.2016-0186OC

Figure 5.

Figure 5.

DCA inhibits TGF-β–induced myofibroblast differentiation. (A) Extracellular lactate measurement in siScr or siHIF-1α HLFs treated with TGF-β or TGF-β + DCA (n = 6 from three biologically independent experiments). HLFs were treated with 5 ng/ml TGF-β and 10 mM DCA for 48 hours. (B) Metabolic flux analysis of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in IPFs treated with 2 ng/ml TGF-β for 48 hours (n = 6 from two biologically independent experiments). IPFs were glucose starved for 2 hours before the assay and then treated sequentially with 10 mM glucose, 10 mM DCA, and 20 mM DCA. (C) Representative immunocytochemistry and quantification of α-SMA in control and DCA-treated IPFs cultured in 21 and 1% O2 and treated with 2 ng/ml TGF-β and 10 or 20 mM DCA for 72 hours (n = 8 from four biologically independent experiments, five to six images were captured per group and normalized to nuclei for quantification). (D) qRT-PCR analysis of α-SMA and calponin1 mRNA expression in control and DCA-treated IPFs cultured in 21 and 1% O2 and treated with 2 ng/ml TGF-β and 10 or 20 mM DCA for 72 hours (n = 6 from three biologically independent experiments). (E) Western blot analysis of p-Smad2/3, p-extracellular signal-regulated kinase (ERK) 1/2, and p-PDH expression in control and DCA-treated HLFs treated with 2 ng/ml TGF-β with or without 10 mM DCA for 1 hour. These results were observed in three independent experiments. N.S. = nonspecific band. (F) Representative immunocytochemistry and quantification of α-SMA in control and DCA-treated HLFs cultured in 1% O2 and treated with 2 ng/ml TGF-β and 10 or 20 mM DCA for 72 hours (n = 6 from three biologically independent experiments, five images were captured per group and normalized to nuclei for quantification). (G) qRT-PCR analysis of α-SMA mRNA expression in control and DCA-treated IPFs cultured in 1% O2 and treated with 2 ng/ml TGF-β and 1–20 mM DCA for 72 hours (n = 6 from three biologically independent experiments). Scale bars: 100 μm. Error bars represent the mean (±SEM). One-way ANOVA with multiple comparison, post hoc.