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. 2011 Apr 7;2(4):e140. doi: 10.1038/cddis.2011.22

Figure 3.

Figure 3

Phenethyl isothiocyanate (PEITC) had similar effects on apoptosis in other human leukemia cell lines as well as primary acute myeloid leukemia (AML) human leukemia cells. U937, Jurkat, and HL-60 cells were treated without or with 8 μM PEITC for 6 h, (a) after which the percentage of apoptotic cells was determined by Annexin V/propidium iodide (PI) staining and flow cytometry as described in Materials and Methods. The values obtained from Annexin V/PI assays represent the mean±standard deviation (S.D.) for three separate experiments. **Values for cells treated with PEITC were significantly increased compared with values obtained for control by Student's t-test; P<0.01. Total cellular extracts were prepared and subjected to western blot analysis using antibodies against poly(ADP-ribose) polymerase (PARP), cleaved-caspase-3 (C-Caspase-3), caspase-8, C-Caspase-9 (b), anti-apoptotic protein Mcl-1 (c), and cell signaling molecules including phospho-Akt, Akt, phospho-Jun N-terminal kinase (JNK), and JNK (d). (e) Blasts from 17 patients with AML were isolated as described in Materials and Methods. After washing and counting, isolated mononuclear cells were treated without or with 8 μM PEITC for 24 h, after which the percentage of apoptotic cells was determined by Annexin V/PI staining and flow cytometry. (f) Total cellular extracts of blasts from two AML patients (P1 and P6) were prepared and subjected to western blot analysis using antibodies against PARP, C-Caspase-3, caspase-8, and C-Caspase-9. (g) Normal peripheral blood mononuclear cells were treated without or with 8 μM PEITC for 24 h, after which the percentage of apoptotic cells was determined by Annexin V/PI staining and flow cytometry as described in Materials and Methods. The values represent the means±S.D. for three replicate determinations. For western blot analysis, each lane was loaded with 30 μg of protein; blots were subsequently stripped and reprobed with antibody against β-actin to ensure equivalent loading. Two additional studies yielded equivalent results