Fig. 1

Effects of vitamin D and bleomycin in A549 cells. a Detection of vitamin D receptor (VDR) in A549 cell extracts. Cells were treated or not with 5 nmol/L vitamin D for 48 h. TBP (Tata box binding protein was used as loading control. KDa: kilodaltons. b RT-qPCR result: expression of CYP24A1 in A549 cells treated for 48 h with 5 nmol/L vitamin D. Values are relative to A549 cells treated with vehicle. c Expression of DSBs induced by bleomycin. Representative micrographs of A549 nuclei harboring DNA damage foci containing TP53BP1, a marker of DSBs, are shown. Right panel: quantification of damaged cells. Scale bar: 5 μm. d Expression of TP53BP1 foci (red dots) in cultures of A549 cells 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. e Quantification of damaged cells (nuclei with TP53BP1 foci) at two and five days after bleomycin shock (12 μg/mL for 6 h). f Quantification of DD foci per nucleus at two and five days after bleomycin shock (12 μg/mL for 6 h). g Senescence-associated β-galactosidase assay (SA-βgal). Representative micrographs of A549 cultures at 48 h post-treatments are shown. Scale bar: 100 μm. Quantification of senescent cells is shown in the right panel. h Detection of γH2AFX, CDKN2A and CDKN1A in A549 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. Cell extracts were obtained at 48 h post-shock. Control cultures were cells treated with bleomycin vehicle (PBS). ACTB was used as loading control. KDa: kilodaltons. Right panel: densitometry analysis of bands; a.u: arbitrary units