Figure 3

Positive and negative modulation of TGFβ1-induced ECM accumulation can be measured via in situ fluorescent staining with Flamingo or incorporation of ¹⁴C-labelled amino acids. Human RPTEC were stimulated with TGFβ1 (30 ng/ml) and the effect of different concentrations of a pan-TGFβ antibody on mature ECM accumulation determined. (A) Decellularised ECM was stained in situ using the Flamingo fluorescent stain, followed by high content image analysis. Data show percent inhibition of the mean target fluorescence intensity for ECM normalised to the PrestoBlue signal as mean ± SD of 4 replicates from a representative of 6 independent experiments. Images representative of a single field are shown. (B) ¹⁴C-labelled amino acid incorporation into deposited ECM was measured. Data show percent inhibition of the radioactivity count for the ECM normalised to the total cell count as mean ± SD of 4 replicates from a representative of 4 independent experiments. The effect of cytokines (10 ng/mL) or nintedanib (1 μM) on ECM accumulation in response to stimulation of RPTEC with 10 ng/mL TGFβ1 was determined via Flamingo in situ staining (C) or incorporation of ¹⁴C-labelled amino acids (D). For each graph, the unstimulated control is shown by the clear bar; grey bars indicate stimulation with 10 ng/mL TGFβ1 whilst striped bars indicate stimulation with 10 ng/mL TGFβ1 in the presence of 0.1% DMSO. Data show mean ± SD from a representative of four independent experiments (replicates shown as individual data points). ****P < 0.0001; **P < 0.01; one way ANOVA Vs. TGFβ1. ⁺⁺⁺⁺P < 0.0001; one way ANOVA Vs. DMSO. ^§p < 0.05; ns, not significant; Student’s t-test Vs. unstimulated.