Figure 2
Figure 2. Vorinostat induces apoptosis in HL cell lines. (A) A representative experiment demonstrating the effect of 2 different concentrations of vorinostat (1 and 5 μM) on apoptosis as determined by the annexin-V binding assay. HL cell lines were incubated with medium or vorinostat (1-5 μM) for 24 or 48 hours before the percentage of dead cells was determined using dual staining with annexin-V and propidium iodide. Vorinostat induces apoptosis in both HL cell lines especially when higher concentrations were used for 48 hours. Percentages of dead cells are shown in each quadrant. (B) Summary results of vorinostst-induced cell death (PI and annexin-V positive cells) from 3 independent experiments. Results after incubations for 24 hours (left panel) and 48 hours (right panel) are shown. Each value is the mean of 3 independent experiments performed in triplicate (± SEM). * denotes a P value of less than .05, and ** denotes a P value of less than .005. (C) HL cell lines were incubated with medium or 5 μM of vorinostat for 6 to 48 hours. Whole cell lysates were examined by Western blot for changes in intracellular proteins. Using this high concentration of vorinostat (5 μM) apoptosis was associated with activation of caspases 8, 9, 3 and PARP cleavage. Consistent with data presented in Figure 2A and B, lower concentrations of vorinostat (1 μM) did not induce caspase activation during the same time frame (Figure S1, available on the Blood website; see the Supplemental Materials link at the top of the online article). (D) Vorinostat-induced cell death of HL cell lines was either partially or completely blocked by the pan-caspase inhibitor Z-VAD-FMK (left panel) or by the caspase 9 inhibitor Z-LEHD-FMK (right panel). HL cell lines were incubated with medium or Z-VAD-FMK (20 μM), Z-LEHD-FMK (20 μM), vorinostat (5 μM), or a combination of vorinostat and Z-VAD-FMK or vorinostat and Z-LEHD-FMK. After 24 hours, the viable cells were counted using a 3-(4,5dimethyltioazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfonyl)-2H-tetrazolium (MTS) assay. Each value represents a mean of 3 independent experiments done in triplicate (± SEM). * denotes a P value of less than .05.

Vorinostat induces apoptosis in HL cell lines. (A) A representative experiment demonstrating the effect of 2 different concentrations of vorinostat (1 and 5 μM) on apoptosis as determined by the annexin-V binding assay. HL cell lines were incubated with medium or vorinostat (1-5 μM) for 24 or 48 hours before the percentage of dead cells was determined using dual staining with annexin-V and propidium iodide. Vorinostat induces apoptosis in both HL cell lines especially when higher concentrations were used for 48 hours. Percentages of dead cells are shown in each quadrant. (B) Summary results of vorinostst-induced cell death (PI and annexin-V positive cells) from 3 independent experiments. Results after incubations for 24 hours (left panel) and 48 hours (right panel) are shown. Each value is the mean of 3 independent experiments performed in triplicate (± SEM). * denotes a P value of less than .05, and ** denotes a P value of less than .005. (C) HL cell lines were incubated with medium or 5 μM of vorinostat for 6 to 48 hours. Whole cell lysates were examined by Western blot for changes in intracellular proteins. Using this high concentration of vorinostat (5 μM) apoptosis was associated with activation of caspases 8, 9, 3 and PARP cleavage. Consistent with data presented in Figure 2A and B, lower concentrations of vorinostat (1 μM) did not induce caspase activation during the same time frame (Figure S1, available on the Blood website; see the Supplemental Materials link at the top of the online article). (D) Vorinostat-induced cell death of HL cell lines was either partially or completely blocked by the pan-caspase inhibitor Z-VAD-FMK (left panel) or by the caspase 9 inhibitor Z-LEHD-FMK (right panel). HL cell lines were incubated with medium or Z-VAD-FMK (20 μM), Z-LEHD-FMK (20 μM), vorinostat (5 μM), or a combination of vorinostat and Z-VAD-FMK or vorinostat and Z-LEHD-FMK. After 24 hours, the viable cells were counted using a 3-(4,5dimethyltioazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfonyl)-2H-tetrazolium (MTS) assay. Each value represents a mean of 3 independent experiments done in triplicate (± SEM). * denotes a P value of less than .05.

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