(A) IMR90 lung fibroblasts were transfected with CBX5 siRNA for 48 hours followed by TGF-β stimulation for 24 hours. qPCR showed that TGF-β–stimulated profibrotic gene expression was significantly impaired in CBX5-silenced fibroblasts compared with those transfected with control siRNA (n = 3). Data are shown as mean ± SEM of 3 independent experiments performed in duplicate. (*P < 0.05, **P < 0.01, ***P < 0.001, by 1-way ANOVA with Turkey’s multiple comparisons test). (B) Western blotting showing that CBX5 knockdown in lung fibroblasts blocked TGF-β–stimulated αSMA expression (representative blot of n = 3). (C) CBX5-silenced lung fibroblasts displayed deficient ECM protein deposition in response to TGF-β, as demonstrated by the immuno-ECM assay. Both fibronectin and collagen I were significantly reduced in CBX5-silenced fibroblasts compared with control cells. Data shown are representative of 3 independent experiments. Results are expressed as mean ± SEM (***P < 0.001 by 2-tailed, paired t test). Scale bar: 10 μm. (D) Scratch assay shows reduced migratory capacity of CBX5-silenced lung fibroblasts in response to TGF-β compared with control cells. Diminished cell migration was significant at 12 hours and remained impaired at 24 hours following TGF-β exposure. Data represent the mean ± SEM from 1 representative experiment performed in triplicate (**P < 0.01 by 2-tailed, paired t test ). Scale bar: 10 μm. (E) qPCR analysis showing that siRNA knockdown of CBX5 in IPF-derived fibroblasts inhibits TGF-β–induced ACTA2 gene expression (n = 3). Data are shown as mean ± SEM of 3 different IPF cell lines (*P < 0.05, ***P < 0.001, by 1-way ANOVA with Turkey’s multiple comparisons test). (F) ACTA2 gene expression is also significantly reduced in CBX5-silenced IPF fibroblasts in absence of TGF-β (n = 4). Data are shown as mean ± SEM of 4 different IPF cell lines (***P < 0.001 by 2-tailed, paired t test). (G) Schematic representation showing interaction of CBX5 with methylated (me) histone 3 on proximal gene promoters.