Fig 1.
The result of drug screening (SCAD inhibitor kits I-IV).
The y-axis represents cell survival (%). Each point represents the result for each compound. HDAC inhibitors are shown in red and labeled with the name of the compounds. The data consists of 4 technical replicates and the values are shown as the mean value ± standard error of the mean.
Table 1.
HDAC inhibitors in the SCAD inhibitor kits, their target HDACs, and anti-proliferative effect against Sora cells (%).
Fig 2.
Growth inhibition of vorinostat on six cUC cells.
The dose-response curves of vorinostat for six cUC cell lines. The data consists of 8 technical replicates and the experiments were repeated independently three times. The values are shown as the mean value ± standard error of the mean.
Fig 3.
Effect of 24 h vorinostat exposure on cell cycle regulation.
A: Representative result of flow cytometric cell cycle analysis using propidium iodide. The x-axis indicates PE intensity (propidium iodide). After data requisition and pre-process by FACSuite, the proportion of each cycle phase was determined by FlowJo software. Red: G0/G1 phase, light blue: S phase, yellow: G2/M phase. B: Summary of the effect of vorinostat treatment on the cell cycle (*P < 0.05, **P < 0.01, t-test, control vs vorinostat).
Fig 4.
Representative results of western blot analysis of H3 acetylation (A-D) and cell cycle-related molecules, p21 and p-Rb (E, F).
Cells were incubated with 1 μM vorinostat for 0.5–6 h (A, B), with 0.1–5 μM vorinostat for 24 h (C, D), and with 1 μM vorinostat for 24 h (E,F). Whole cell lysates were analyzed by western blot analysis.
Fig 5.
The effect of vorinostat in cUC xenograft model.
Changes in tumor volume (A, p = NS by ANOVA, control vs. vorinostat), tumor volume after harvested on day 29 (B, *p < 0.05 by student-t test, control vs. vorinostat) and the representative macroscopic appearance of xenografted mice on day 29. (C, scale bar = 1 cm).
Fig 6.
Immunohistochemistry of cUC tissues and correlation between percentages of acetylated cells and prognosis.
A: The percentage of acetyl-histone H3K9 positive cells in normal bladder tissues and cUC tissues (*** P < 0.001, Mann–Whitney U test, normal bladder tissues vs cUC tissues). B: Representative images of immunohistochemical staining of acetyl-histone H3K9 in normal bladder tissues and cUC tissues (high acetylation group and low acetylation group). C: Kaplan–Meier survival analysis of PFS (C) and OS (D) in cUC cases (log-rank test, high acetylation group vs low acetylation group). Two cases (OS) and four cases (PFS) were excluded because of perioperative death and due to the unavailability of data.