Figure 6
K Ca 3.1 channel inhibition reduces constitutive HLMF αSMA expression. (A) The percentage of cells expressing αSMA was dose-dependently decreased by TRAM-34 in NFC (n = 3) and IPF (n = 3)-derived HLMFs (a minimum of 10 random cells were measured in one field for each donor, data for NFC and IPF pooled for statistical analysis). (B) Similarly, αSMA expression was dose-dependently attenuated by ICA-17043 in NFC (n = 5) and IPF (n = 3)-derived HLMFs (data pooled for statistical analysis). No differences were evident between NFC and IPF-derived cells. (C) Representative immunofluorescent images illustrating decreased αSMA expression in the actin filaments and cytoplasm following KCa3.1 inhibition with TRAM-34 (20 nM and 200 nM) and ICA-17043 (10 nM and 100 nM). (D) Two structurally related molecules without channel blocking activity, TRAM-85 and TRAM-7, did not reduce constitutive αSMA expression in NFC (n = 2) and IPF (n = 2)-derived HLMFs (data pooled, P < 0.999 and p = 0.1244 respectively). (E and F) The mean fluorescent intensity (MFI) of constitutive αSMA expression was also assessed by flow cytometry. TRAM-34 (200 nM) (E), and ICA-17043 (100 nM) (F), again reduced αSMA expression significantly in NFC (n = 4) and IPF (n = 4)-derived HLMFs (data pooled for statistical analysis). Results are represented as mean ± SEM ***P < 0.0001 (2 way ANOVA corrected by Dunnetts multiple comparison test) or median ± IQR #P < 0.05 and ##P < 0.01 (Wilcoxon signed rank test).