Figure 3
Total and nuclear Smad2 and Smad3 expression is significantly higher in IPF-derived HLMFs. (A) Smad2 mRNA expression was examined by qRT-PCR in NFC (n = 4) and IPF (n = 4) myofibroblasts. IPF-derived HLMFs had significantly higher Smad2 mRNA at baseline in comparison to NFC-derived cells. (B) Similarly, Smad3 mRNA expression was significantly higher in IPF-derived HLMFs (n = 4) in comparison to NFC-derived cells (n = 4). (C) Smad3 mRNA expression was increased compared to Smad2 mRNA expression in both NFC and IPF-derived HLMFs. (D and E) Quantification of Western blot analysis showed that total Smad2/3 protein was increased in IPF-derived HLMFs in comparison to NFC-derived cells. Results were normalised to β-actin. (F) The proportion of total Smad2/3 nuclear staining to whole cell staining was measured in at least 10 cells in HLMFs from both NFC (n = 7) and IPF (n = 9) donors. IPF-derived HLMFs had a significantly higher proportion of Smad2/3 located in the nucleus compared to NFC-derived cells. (G) A representative illustration of total Smad2/3 expression assessed by immunofluorescence. (H) Representative Western blot analysis showing total Smad2/3 protein within the nucleus and cytoplasm of IPF and NFC-derived cells. TBP is localized to nuclear enriched fractions relative to cytoplasmic enriched fractions, similarly β-actin is localized to cytoplasmic enriched fractions relative to nuclear enriched fractions, N = nucleus, C = cytoplasm. Detected bands were at the correct molecular weight of 52 and 60 kDa for total Smad2 and Smad3 respectively, 38 kDa for TBP and 43 kDa for β-actin (I) Quantification of Western blot analysis confirmed that total Smad2/3 in the nuclear enriched fraction is significantly increased in IPF cells (n = 3) in comparison to NFC cells (n = 3). Results were normalized to TBP. (J) Similarly, total Smad2/3 in the cytoplasmic enriched fraction was increased in IPF cells (n = 3) in comparison to NFC-derived cells (n = 3). Results were normalized to β-actin. Results are represented as mean ± IQR #P < 0.05 (Mann Whitney) or mean ± SEM *P = 0.05 (Un-paired t-test).