Fig. 3

Effects of vitamin D and bleomycin in human myofibroblasts. a Representative micrographs of a typical myofibroblasts culture. Scale bar: 100 μm. b Accumulation of population doubling levels (PDLs) during ten consecutive passages. c Detection of vitamin D receptor (VDR) in myofibroblasts extracts. TUBA1A (tubulin α was used as loading control. KDa: kilodaltons. d RT-qPCR result: expression of CYP24A1 in myofibroblasts cultures treated with 5 nmol/L vitamin D. Values are relative to cells treated with vehicle. e Expression of TP53BP1 foci (red dots) in cultures of myofibroblasts pretreated with 5 nmol/L vitamin D for 2 h, subjected to a bleomycin shock (12 μg/mL for 6 h) and then treated with 5 nmol/L vitamin D or its vehicle. Representative micrographs taken at 48 h post-shock are shown. Scale bar: 20 μm. Right panel: quantification of damaged cells (nuclei with TP53BP1 foci). f Quantification of the senescence levels (% of SA-βgal⁺ cells). Myofibroblasts were pretreated with 5 nmol/L vitamin D for 2 h, subjected to a bleomycin shock (12 μg/mL for 6 h) and analyzed at 48 h post-shock. PBS is the bleomycin vehicle; veh.: vitamin D vehicle. g Detection of γH2AFX, CDKN2A and CDKN1A in myofibroblasts cell extracts. Cells were pretreated with 5 nmol/L vitamin D for 2 h, subjected to a bleomycin shock (12 μg/mL for 6 h) and then treated with 5 nmol/L vitamin D or its vehicle. PBS is the bleomycin vehicle; veh.: vitamin D vehicle. ACTB was used as loading control. KDa: kilodaltons