Figure 6. The HDAC inhibitor SAHA decreases Notch3 stability in urothelial cancer cells.

(A) Notch3 protein levels after treatment with SAHA at different concentrations or DMSO (vehicle control), determined by western blot. (B) Colony formation efficiency was assessed for T24 cells in presence of SAHA at different concentrations or DMSO (vehicle control). (C) T24 cells were treated with 10 μM SAHA or the vehicle alone. Total protein samples were subjected to immunoprecipitation (IP) with anti-Notch3 followed by western blot using anti-acetyl K and anti-Notch3 antibodies. (D) Ki-67 levels in T24 cells after treatment with 10 μM SAHA or the vehicle alone, determined by immunofluorescence. Representative photomicrographs are presented from three independent experiments. Scale bar, 50 μm. (E) Cell cycle analysis was performed on T24 cells after treatment with 10 μM SAHA or the vehicle alone for 2 days. Then, cells were stained with propidium iodide and analyzed by flow cytometry. (F) T24 cells were treated with the proteasome inhibitor MG132 (10 μmol/L). Notch3 protein levels were analyzed by western blot. *P < 0.05 vs. the other groups. (G) After T24 cell treatment with cycloheximide (CHX, 100 μmol/L) for the indicated time intervals, Notch3 protein amounts were analyzed by western blot. *P < 0.05 vs. control. (H) Ubiquitinated Notch3 was purified from MG132-treated T24 cells. Whole-cell lysates, from T24 cells treated with SAHA (18 h, 10 μM) or the vehicle alone, were immunoprecipitated with anti-Notch3 antibody followed by western blot for ubiquitin detection.