FIGURE 6.

Generation and verification of an Acta2 coupled BFP reporter cell line. (A,B) Schematic representation of 10-4A cell transfection. Cells were transfected with a plasmid containing Cas9 and the sgRNA binding at the desired gene (exon 10) and a donor plasmid. The donor plasmid contained a BFP flanked by a self-cleaving T2A cassette, a nuclear localization sequence and two 500 bp homologous arms of the sgRNA cutting site. After cell transfection, Acta2 gene expression was induced by addition of 5 ng/ml TGF- β1 and cells were FACS sorted for BFP. Semi-quantitative RT-PCR analysis of Acta2 (C), Col1a1 (D), and Plin2 (E) expression in 10-4A and 10-4A^BFP cells with and without administration of 5 ng/ml TGF-β1 on a soft PDMS substrate, respectively. Values are means from five individual culture experiments. The respective time points were tested with the non-parametric Mann–Whitney-U-Test. Statistical significance is indicated as follows: p-values < 0.05: *, p-values < 0.01: **. Box plots show data as median values, the boxes represent percentiles, the whiskers indicate the minimum/maximum. (F) Immunofluorescence staining of 10-4A^BFP cells seeded on PDMS with and without administration of 5 ng/ml TGF-β1 directly after adherence (d0) or 7 days post-seeding. Cells were monitored for BFP and stained for the mesenchymal marker vimentin (green) and the pro-fibrotic protein αSMA (red). Scale bar = 50 μm. (G) Correlation of the BFP and αSMA signal intensity within individual cells 7 days post seeding. N = 48 cells. A linear regression line and the corresponding indicator R and p-value show the linear dependency of BFP and αSMA signal.