Fig. 1: GO-induced apoptotic signaling involves caspase-2 processing.

A HL60 AML cells were treated with GO 100 ng/ml or 1000 ng/ml for 24 h or 48 h. Cell viability was assessed by trypan blue exclusion staining. Data shown is the average of three independent experiments with bars representing SD. B Apoptotic associated fragmentation of cell nuclei was analyzed in samples treated as in (A) by staining with DAPI and counting in fluorescence microscope. Data shown is the percentage of cells showing apoptotic fragmentation of DNA in nucleus out of 200 cells counted. Data given is the mean of three biological replicates with bars representing SD. C DNA DSB rejoining was assessed in HL60 cells at indicated time points after treatment with GO (left panel), calicheamicin or etoposide (right panel) using PFGE. The amount of DNA DSBs is presented as fold fragmented DNA (size < 5.7 Mbp) relative to untreated cells. D Full-length and processed caspase-2 (51 kDa and 35 kDa, respectively) were analyzed in HL60 cells following GO treatment for 24 h. Fold calculation were comparing the 35 kDa cleaved fragment in relation to untreated cell after adjustment to GAPDH expression. Figure shown is representative of two independent biological replicates. E Full-length caspase-2 expression was analyzed by western blot in primary mononuclear cells from AML patients (pat#1, #12, and #13; Table 1) treated ex vivo for 24 h with indicated GO doses. Expression of full-length caspase-2 was quantified in relation to each patient untreated sample set to 1 after normalization to the GAPDH loading control.