Figure 4. Caspase-7 Cleaves HDAC3 at Asp391.

(A–D and F–K) Immunoblotting analyses with the indicated antibodies.

(A) 3Y1 cells were pretreated for 30 min with or without Z-VAD-FMK (75 μM) before sorbitol treatment for 6 h.

(B) In vitro caspase cleavage assay with mixed purified active His-caspase-1, −2, −3, −4, or −7 and purified His-HDAC3 or immunoprecipitated PARP from 293T cell lysate.

(C) 3Y1 cells were treated with or without sorbitol for 6 h.

(D) 3Y1 cells were pretreated with caspase-3 inhibitor V (25 μM) or caspase-3/7 inhibitor I (20 μM) for 30 min before sorbitol stimulation for 6 h.

(E) 293T cells stably expressing pc-Jun-Luc were pretreated with or without Z-VAD-FMK (75 μM) or caspase-3/7 inhibitor I (20 μM) before sorbitol stimulation for 6 h, and luciferase activity was determined. The relative levels of luciferase activity were normalized to the levels of untreated cells and to the levels of luciferase activity of the Renilla control plasmid. Data represent the means ± SD of three independent experiments.

(F) Cell lysates were prepared from 3Y1 cells expressing pRS control shRNA or pRS caspase-7 shRNA.

(G–I) 3Y1 cells expressing the indicated shRNA (G), DT40 cells and DT40 caspase-7^−/− cells (H), and MEK2^+/+ and MEK2^−/− cells (I) were treated with or without sorbitol for 6 h.

(J) In vitro caspase cleavage assay with mixed purified His-caspase-7 and WT His-HDAC3 or the D391A mutant.

(K) 293T cells transiently expressing WT FLAG-HDAC3 or the D391A mutant were treated with or without sorbitol for 6 h.