Fig. 2
Combination of domatinostat and gemcitabine/taxol induces apoptosis and reduces clonogenic capacity in PDAC cells. A. Flow cytometry analysis of Annexin-V shows the outcome of the induced chemosensitivity in PANC1, PANC28, ASPC1 and BjhTERT (human-derived fibroblast) cells when domatinostat (0.5 μM) is combined with gemcitabine/taxol (GT) (IC50 at 96 h) for 24 h. B. Apoptosis, evaluated by PARP cleavage induction. In PANC1, PANC28, ASPC1 cells PARP cleavage is induced when the cells are treated with domatinostat (0.5 μM) plus GT (IC50 at 96 h) for 48 h. C-D-E. Clonogenic assay shows the long-term effects of combination treatment domatinostat (0.1 μM) plus GT (IC10 at 96 h) in PDAC cell lines collected 10–15 days after treatment. A one well picture in a representative experiment is shown for each treatment; bar graphs show the numbers of colonies for each condition (mean ± SD of 2 or more separate experiments each one with technical triplicate). F. Limiting dilution assay performed on PDAC cells, untreated or treated for 24 h with domatinostat (0.1 μM) plus GT (IC10 at 96 h) and plated in ultra-low 96-well without additional treatment for three weeks. Clonal frequency was evaluated with the Extreme Limiting Dilution Analysis ‘limdil’ function as described in Material and Methods section. Data are mean ± SD; n = 3 independent experiments, analyzed using two way ANOVA test. **P ≤ 0.01, ***P ≤ 0.001