Fig. 1

Long-term, dose- and time-dependent effects of sulforaphane on murine NIH/3t3 fibroblasts. A-B 1,000 NIH/3t3 cells were treated with increasing concentration of sulforaphane (SFN; 1.5 – 48 μM) for 3 (A) or 7 days (B). Treatment with 0.1% (v/v) DMSO was also performed as a vehicle control. The relative amount of viable cells was measured by MTT assay. Absorbance data are normalized to the control group (Not treated, NT; 0 μM SFN) and represent the mean ± SEM of three independent experiments, each one performed in triplicate replicates. C Optimized procedure for SFN treatment of NIH/3t3 cells for exosome extraction: 70,000 NIH/3t3 are cultured on 100 mm cell-culture dishes and treated with ± 3 μM SFN in complete exosome-free culture medium. The treatment is repeated every 3 days and cells are maintained for a total of 7 days; conditioned medium is collected at each medium change (day 3 and 6) and at the end of treatment (day 7). D–F 70,000 NIH/3t3 cells were treated with ± 3 μM SFN for 7 days in 100 mm cell-culture dishes. Total cell lysate was obtained and the expression of acetylated (Ac) H4 / total H4 histone (D), Ac H3/total H3 histone (E) and Nrf2/β-tubulin (F) was measured by Western blotting. A representative blot is shown for each protein. Densitometric data are normalized to the loading control (β-Tubulin), expressed as relative to the control group (not treated, NT) and represent the mean ± SEM of three independent experiments, each one performed in triplicate replicates. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs NT