Cells were treated with defined concentra tions of a belinostat stock dissolved in DMSO. Control cells were grown fol lowing a similar protocol in medium supplemented with equivalent volumes of PBS. MTT assay for cell proliferation A 3 2. 5 diphenyltetrazolium bromide assay was used to assess cell proliferation Oligomycin A price reflected by metabolic activity of the cells. The cells were seeded in 96 well plates at a density of 5000 cells well in 250 ul of complete medium. After the cells became ad herent, they were exposed to belinostat for 48 h. Cells exposed to PBS served as controls. After the indicated times, 10 ul of MTT were added to each well and incubated for 4 h. After incubation, the culture medium was aspirated and the plates were dried by inversion for about 15 min.

Subsequently, the formazan products were solubilised with acidic isopropanol and the optical density was measured at 570 nm with a Mul tiscan EX plate reader. All assays were performed in triplicate. Proliferation in the control group was set as 100%. Immunoblotting After 24 h treatment with Belinostat, cells were washed twice with ice cold PBS before lysis with SDS lysis buffer. Protein concentration was determined by BCA protein assay. Cell lysates were separated on a 15% SDS poly acrylamide gel and electroblotted on PVDF membrane. Membranes were then incubated in blocking solution, followed by over night incubation at 4 C with anti acH4, CHIP Grade antibody at a di lution of 1 75,000, or rabbit polyclonal anti p21Cip1 Waf1 at a 1 200 dilution.

The mem branes were then washed in TTBS and incubated with secondary antibodies POD conjugated donkey anti rabbit at 1 150,000 dilution or POD conjugated goat anti rabbit IgG at 1 2000 dilution. Antibody detection was performed using an enhanced chemiluminescence reaction. Equal lane loading was confirmed using anti actin antibody. The actin sig nal obtained after incubation with anti actin antibody on the same membrane was used as an internal control, in addition to loading all lanes with the same amount of protein. All assays were performed in triplicate. For the semiquantitative analysis of the immunoblots, densitometry using the ImageJ program was carried out, and the signal intensity of acH4 or p21Cip1 Waf1 expres sion was normalised to its corresponding signal intensity of actin.

Induction and analysis of cell death by flow cytometry Belinostat was diluted in phosphate Batimastat buffered saline to a final concentration ranging from 100 to 1000 nM, according to the concentrations indicated in each experiment. Gemcitabine was applied at a final concentra tion of 0. 01 mM in PBS. All cells were treated and culti vated under the same conditions, and exposed to the drugs 24 h before the experiments. The viability of PDAC cells after exposure to belinostat and or gemcitabine was analysed using annexin V propi dium iodide staining.