Figure 5
Azathioprine delays FAPs growth without inducing apoptosis. (A) Experimental plan for EdU labelling of in vitro cultured mdx FAPs. (B) The average ratio of EdU-positive FAPs over the total cell number in each condition. (C) Time course of the average number of cells per field. (D) The average ratio of ORO-positive cells over the total cells in each condition. (E) Representative TUNEL assay images of mdx FAPs immunostained 20 and 48 hours after AZA (0, 1, 10, 25 μM) and TSA (50 nM) treatment. A sample stained with the label solution alone and a sample pre-treated with DNase I were used as negative and positive control, respectively. Nuclei (blue) were stained with Hoechst 33342. (F) The bar plot represents the average of the ratio between TUNEL-positive dots and the number of cells in each field for each experimental condition. The statistical significance, was estimated by two way ANOVA and defined as *p < 0.05; **p < 0.01; ***p < 0.001. (G) Representative immunoblot in which for each sample 15 μg of protein lysate was electrophoresed on a 15%® SDS-PAGE and the protein levels of the cleaved and the total form of Caspase-3 were simultaneously revealed with a specific antibody. Actin serves as loading control. Full-length blots are represented in Supplementary Fig. S9. (H) The bar plot represents the densitometric analysis of the ratio between the cleaved Caspase-3 over the total signal of caspase-3. 1 μM staurosporine (STA) was used as positive control. All experimental values are represented as means of at least three independent experiments ± SEM and the statistical significance, was estimated by one way ANOVA and defined as *p < 0.05; **p < 0.01; ***p < 0.001. Scale bar: (E) 100 μm.
