Fig. 1.

HDAC inhibition reduces Notch3 levels and signaling in T-ALL cells. a T-ALL cells (DND 41, MOLT3, and Jurkat) were treated with TSA (0.5 µM) or solvent (DMSO) for 16 h and protein levels analyzed by western blot. Actin was used as a loading control and tubulin acetylation and c-Myb levels as markers of HDAC inhibition. b TSA reduces Notch3 surface expression in T-ALL cells. DND 41 and MOLT3 cells treated with TSA or DMSO for 16 h were stained with PE anti-human Notch3 (anti-N3 Ab) or with isotype control antibody and analyzed by flow cytometry. One representative experiment of three performed is shown. Histogram reports fluorescence mean intensity (FMI) ± SD of three independent experiments (**P < 0.01; *P < 0.05). c TSA reduces Notch3 expression in PDX-derived T-ALL cells. T-ALL cells obtained from the spleen of xenografted mice were treated in vitro with TSA for 16 h and protein levels were analyzed by western blot. d-h Effects of TSA on Notch3 target genes and on Notch transcript levels. T-ALL samples, including both cell lines (d, f, g) and PDX T-ALL cells (e, h), were treated with TSA or Givinostat (GIV) (2 µM) for 16 h and mRNA levels of NOTCH3, c-MYB, or Notch target transcripts (pTα, CR2, DTX-1) were analyzed by qRT-PCR. n.d.: not detectable. Statistically significant differences are indicated (*P < 0.05, **P < 0.01, ***P < 0.001, mean ± SD of three independent experiments). Expression data are normalized to DMSO samples