Fig. 6.

Pharmacologic or genetic HDAC6 inhibition is associated with reduced Notch3 protein levels in vivo and impaired tumor growth. a Outline of treatment. PD-TALL12 xenografted NOD/SCID mice were randomized to receive either Givinostat (25 mg/kg) or vehicle i.p. and sacrificed 16 h after treatment. b Leukemic cells were recovered from the spleen of PD-TALL12 mice and Notch3 FL and acetylated α-tubulin protein levels analyzed by WB. Numbers indicate results of densitometric analysis of Notch3 FL bands normalized to Actin. c Columns report the mean values ± SD of Notch3 to actin ratios (densitometric analysis) in control and treated mice (***P < 0.001). d TALL1 cells were serially transduced with a lentiviral vector encoding the Firefly luciferase gene (fLUC) and with lentiviral vectors expressing either a scramble shRNA or an HDAC6-specific shRNA (shHDAC6 #1). Cells were i.v. injected in NOD/SCID mice (2.5 × 10⁶ cells/mouse, n = 5 mice/group) and tumor growth was monitored by optical imaging (e, f). Representative images (e) and quantitative analysis (f) of luciferase activity at day 35 from TALL1 cells injection. Statistically significant differences in average radiance in the two groups of samples are indicated (*P < 0.05, mean ± SD, n = 5 mice/group). g Flow cytometric analysis of CD7⁺ cells in the BM and spleen of shRNA and shHDAC6#1 mice at sacrifice (41 days) (**P < 0.01, mean ± SD, n = 5 mice/group). h Apoptosis was analyzed by caspase 3–7 assay in CD7⁺ cells sorted from the spleen of mice at sacrifice (*P < 0.05, mean ± SD, n = 5 mice/group). i After sorting, T-ALL cells obtained from different mice were pooled and Notch3 FL protein levels analyzed by western blotting