Fig. 4

Halofuginone inhibits pro-angiogenic mediators in NB4 cells. a Phospho-SMAD2 identification assessed by ELISA in NB4 cells treated with 50 and 200 ng/mL of HF for 6 and 24 hours hours and TGF-β (1ng/mL) was added to the cultures 1 hour before. b Concentration of VEGF determined by CBA in the supernatant of NB4 cells treated with 50 and 200 ng/mL doses of HF during 72 hours. CTRL: non-treated cells. c, d ChIP assay using antibodies to P-Smad2 and Smad2/3. NB4 cells were treated with 200 ng/mL of HF for 6 hours, or treated with 200 ng/mL of HF for 6 hours and stimulated with TGF-β (1ng/mL) in the last hour, or only stimulated with TGF-β. Graphics showing a PCR amplification of ChIP products using a series of primer pairs covering the upstream and downstream the transcription start site (TSS) of Vegf. Data is represented as fold enrichment to IgG control antibody. **P < 0.001, ***P < 0.0001; (one-way ANOVA). Data are representative of two a and three b) independent experiments (error bars, s.e.m.)