Figure 3.
Drug-like OGA inhibitor KCZ cleaves Bid to its truncated active form tBid. (A) Analysis of apoptosis signaling proteins in response to BTZ and KCZ co-treatment. MCL Jeko-1 cells were treated with various concentrations of BTZ (0–7 nM) and KCZ (25–75 μM) for 24 h and analyzed for key apoptosis regulatory proteins, including Bcl-2, Bax, p53, p73, Mcl-1, caveolin-1 (Cav-1), Bid and caspase-9 (C9) using Western blotting. Blots were reprobed with anti-β-actin antibody to confirm equal loading of the samples. (B) KCZ potentiates caspase-8 activation and Bid cleavage in BTZ-treated cells. Cells were treated with various concentrations of BTZ and KCZ for 24 h and the levels of pro- and activated caspase-8 (C8) as well as Bid and tBid were determined by Western blotting. Quantitative analysis of Bid and tBid by densitometry is shown. Data are mean ± s.d. (n=4). *P < 0.05 vs. non-treated cells; two-sided Student’s t-test. #P < 0.05 vs. BTZ-treated cells; two-sided Student’s t-test. (C) C8 activity was evaluated in the cells treated with BTZ (6 nM) and increasing concentrations of KCZ (0–75 μM) for 16 h using the fluorometric substrate IETD-AFC. Data are mean ± s.d. (n=3). Data are mean ± s.d. (n=3). *P < 0.05 vs. non-treated cells; two-sided Student’s t-test. #P < 0.05 vs. BTZ-treated cells; two-sided Student’s t-test.