PMC

Cell cycle analysis of PC3-PSMA cells treated with 0.5% DMSO, 3.3 μM Entinostat, and 33 μM Entinostat (n = 3); results from one representative experiment are shown. The y-axis indicates the number of cells with the specific fluorescence intensity shown on the x-axis.

Enhancement of EGFP expression by Entinostat in PC3 and PC3-PSMA human prostate cancer cells at 24 and 48 h post-transfection with PEI (polymer:plasmid DNA mass ratio of 1:1). These representative images are consistent with n = 3 independent experiments.

Effect of Entinostat on luciferase expression (left) and cell viability (right) in PC3-PSMA cells, following transfections with PEI in serum (10% FBS)-containing media, that is, SCM or serum free media, that is, SFM (n = 3). Data shown indicate mean values + one standard deviation. Asterisks (*) denote significant differences relative to the polyplex control (0 μM Entinostat).

DNase accessibility data normalized to DMSO treatment (n = 3) in PC3-PSMA cells. The y-axis indicates fractional DNase accessibility relative to DMSO-treated cells. Data shown indicate mean values ± one standard deviation. For all four regions, as indicated by primer pairs 1–4 in the promoter/gene map, the difference in DNase accessibility between DNA harvested from DMSO and Entinostat (33 μM) treated cells was not found to be statistically significant (P < 0.05 threshold, Student’s t-test).

Effect of Entinostat on global protein expression (as measured via BCA assay) and transgene (luciferase) expression in PC3-PSMA cells transfected with 10:1 (w/w) 1,4C-1,4Bis pGL3 polyplexes in serum free-media, that is, SFM (n = 3). The protein/cell and RLU/cell reported for Entinostat in this graph are normalized with those observed for the DMSO (i.e., vehicle control in absence of Entinostat). Data shown indicate mean values ± one standard deviation. Asterisks (*) denote significant increase in RLU/cell relative to protein/cell with 33 mM Entinostat treatment.

Relative luciferase expression (left column) and cell viability (right column) in PC3 human prostate cancer (top row), PC3-PSMA human prostate cancer (middle row), and MB49 murine bladder cancer (bottom row) cells. Asterisks (*) denote statistically significant (P < 0.05) enhancement of luciferase expression or reduction of viability relative to the corresponding polyplex controls. Data shown indicate mean values ± one standard deviation. Note: luminescence and viability were not measured at 100 μM Entinostat in MB49 with PA8 polymer.

Normalized plasmid content in the nuclear fraction, indicating the relative amount of exogenously delivered plasmid DNA present in the nucleus following Entinostat treatment relative to vehicle control (DMSO) treatment (n = 3). Data are reported for (A) PC3-PSMA (33 μM Entinostat) and (B) PC3 cells (10 μM Entinostat), and are represented as mean ± one standard deviation. Asterisks (*) denote P < 0.05 comparing pGL3 levels in nuclear fraction in Entinostat-treated cells relative to DMSO treated cells (Student’s t-test). The base pairs are labeled such that position 1 is the first base pair amplified by our primers. The numbering system was defined for convenience, and is not related to that provided by the vendor (Promega).