F0313

HLMFs were grown on 8-well chamber slides and serum-starved for 24 h prior to the experiment. The cells were then stimulated with TGFβ1 (10 ng/ml) in the presence of either 0.1% DMSO control, TRAM-34 (200 nM), ICA-17043 (100 nM), TRAM-85 (200nM) or Ca²⁺-free media. After 1 h, the cells were fixed with methanol for 20 min on ice, blocked using 3% BSA for 1 h and immunostained using rabbit monoclonal anti-Smad2/Smad3 (0.174 μg/ml, Cell Signalling). Secondary antibody labelled with FITC (F0313, Dako) was applied, and the cells counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, St. Louis, MO, USA). The cells were mounted with fluorescent mounting medium and cover-slipped.
Original images were captured on an epifluorescent microscope (Olympus BX50, Olympus UK Ltd., Southend-on-sea, Essex, UK); grey scale intensity was examined using Cell F imaging software (Olympus UK Ltd., Essex, UK). Matched exposures were used for isotype controls. The intensity of nuclear Smad2/3 staining was quantified by measuring the grey scale intensity of DAPI positive nuclei with a minimum of 10 random cells measured in one field for each condition.

Cells were grown on T25 flasks and serum-starved for 24 hours prior to the experiment. The myofibroblasts were incubated for 24 hours in the presence of 0.1% DMSO control, TRAM-34 (200 nM) or ICA-17043 (100 nM). Cells were detached using 0.1% trypsin/0.1% EDTA, washed then fixed and permeabilised in 4% paraformaldehyde plus 0.1% saponin (Sigma) respectively for 20 minutes on ice. Myofibroblasts were labelled with FITC-conjugated mouse monoclonal anti-α-smooth muscle actin (sigma) or isotype control FITC-conjugated mouse IgG_2a. Secondary antibodies labelled with FITC (F0313, Dako) were applied. Analysis was performed using single colour flow cytometry on a FACScan (BD, UK).