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. 2010 Jan 29;37(2):159–171. doi: 10.1016/j.molcel.2009.12.023

Figure 3.

Figure 3

HtrA2 Is Required for the Stimulation of WT1 Cleavage following Treatment with Cytotoxic Drugs

(A) Mouse embryonic M15 cells were treated for 20 hr with 10, 30, and 100 μM Etoposide and analyzed by immunoblotting with WT1 antibodies (C-ter). The 20 kDa cleavage product is indicated by an arrow. Nonspecific bands are indicated by asterisks.

(B) Human K562 cells were incubated with the HtrA2-specific inhibitor UCF-101 (15, 30, and 45 μM) for 30 min before stimulation with 100 μM Etoposide for 24 hr and were analyzed as in (A). Nonspecific bands are indicated by asterisks.

(C) U2OS (left) or H1299 cells (right) were transfected with control siRNA or with two different HtrA2-specific siRNAs. HtrA2 knockdown was confirmed by immunoblotting. At 60 hr after transfection, the cells were stimulated with 0.5 μM Staurosporine or 5 ng/ml Anisomycin for 8 hr and analyzed by immunoblot with the antibodies indicated. The lower immunoblot for the U2OS cells used a different anti-WT1 (C-Ter) antibody that is more sensitive, and the 20 kDa C-terminal proteolytic fragment is indicated.

(D) Untreated M15 cells or M15 cells that had been treated with 30 μM etoposide for the time points indicated were fractionated to produce nuclear (Nu) and cytoplasmic (Cyt) fractions. The samples were immunoblotted with the antibodies indicated.