Fig. 4

‘Scar-in-a-jar’ high content screening: compound potency (pIC₅₀) determination. a Representative immunocytochemical image of 96-well plate after 72 h ‘scar-in-a-jar’ assay. Wells B1, C1, D1, E12, F12, G12 were treated with 0.1% DMSO (vehicle) in ficoll media, and wells E1, F1, G1, B12, C12, D12 were treated with vehicle in the presence of TGFβ₁ [1 ng/ml] in ficoll media. Positive controls, SB-525334 (Alk5 inhibitor), PGE₂ and CZ415 (mTOR inhibitor) were assayed in a 10-point concentration response in duplicate (0.1% DMSO). Rows A and H were filled with PBS to minimise plate effect. Wells were stained for collagen type I, and Hoechst to visualise ECM deposition and cell count. Images were acquired using a CellInsight HCS microscope at 10x magnification. Scale bars represent 200 μm. b-d Representative screening data from assay positive controls (b) SB-525334 (Alk5 inhibitor), (c) PGE₂ and (d) CZ415 (mTOR inhibitor). Graphs indicate the quantification of collagen type I deposition (Alexa488) and cell count (nuclei count, Hoechst) as determined by immunocytochemical analysis of IPF lung fibroblasts after 72 h. Vehicle (Veh) data points represent basal collagen deposition in the presence of 0.1% DMSO. Data points were plotted from mean ± SEM from 10-point concentration response curves in duplicate (a). **P ≤ 0.01, ***P ≤ 0.001 one-way ANOVA. 10x magnification