Inhibition of the K_Ca3.1 channel attenuates features of TGFβ1-dependent EMT.

(A) Cell elongation, quantified by a ratio of cell length:cell width of vimentin-stained BEAS-2B cells after 72 h, was increased by TGFβ1 or TGFβ1+DMSO compared to untreated cells (0.1% PBS/BSA; *P < 0.001). Pre-treatment of BEAS-2B cells with 200 nM TRAM-34 (#P < 0.001) or 100 nM ICA-17043 (##P = 0.027) significantly inhibited TGFβ1-induced cell elongation compared to TGFβ1+DMSO control. In contrast, TRAM-7, an inactive analog of TRAM-34, did not inhibit TGFβ1-induced cell elongation (**P = 0.022 compared to TRAM-34). Data are presented as mean ± SEM from 6 individual experiments. (B) BEAS-2B cells treated with 10 ng/ml TGFβ1 or TGFβ1+DMSO for 72 h exhibited a loss of E-cadherin expression in comparison to untreated cells (0.1% PBS/BSA; *P < 0.001, **P = 0.001). Pre-treatment with TRAM-34 (200 nM; #P = 0.013) or ICA-17043 (100 nM; ##P = 0.002) attenuated the TGFβ1-dependent loss of E-cadherin compared to TGFβ1+DMSO. TRAM-7 (200 nM) did not prevent TGFβ1-induced loss of E-cadherin immunostaining (***P = 0.014 compared to TRAM-34). Data are presented as mean ± SEM from 6 individual experiments. (C) BEAS-2B cells treated with 10 ng/ml TGFβ1 or 10 ng/ml TGFβ1+DMSO control for 72 h displayed an increase in collagen-1 expression in comparison to untreated cells (0.1% PBS/BSA) (*P = 0.005; **P = 0.028 respectively). However, pre-treatment with TRAM-34 (200 nM; #P = 0.021) or ICA-17043 (100 nM; ##P = 0.024) significantly inhibited this effect compared to TGFβ1+DMSO. TRAM-7 (200 nM) did not inhibit TGFβ1-induced upregulation of collagen-1 immunostaining (***P = 0.021 compared to ICA-17043). Data are presented as mean ± SEM from 5 individual experiments.