Figure 3
Figure 3. ST1926 induces partially caspase-dependent apoptosis and results in elevated levels of p53 and γ-H2AX proteins and in reduced Tax protein levels. (A) ST1926 causes PARP and caspase 3 cleavage in HuT-102, MT-2, Jurkat, and MOLT-4 cells. Total sodium dodecyl sulfate protein lysates (50 µg/lane) were prepared after treating malignant T cells with 1 μM ST1926 for up to 48 hours. Lysates were immunoblotted against PARP and caspase-3 antibodies. Arrows indicate cleaved PARP and caspase 3. (B) Effects of the general caspase inhibitor z-VAD on ST1926-induced growth inhibition of HuT-102, MT-2, Jurkat, and MOLT-4 cells. Cells were pretreated for 2 hours with 100 μM z-VAD, followed by 24-hour treatment with 1 μM ST1926. Cell viability was determined in quadruplicate wells by the trypan blue dye exclusion and expressed as percentage of control. Results are an average of 2 independent experiments ± SE. *Statistically significant (P < .05) differences (independent t test). (C) ST1926 upregulates p53 and its phosphorylated form (P-p53) in malignant T cells. HuT-102, MT-2, Jurkat, and MOLT-4 cells were treated with 1 μM ST1926 for up to 48 hours and immunoblotted against p53 and P-p53 antibodies. Similar trends in protein levels were observed in 2 independent experiments. Bands were quantified, and results are expressed relative to control cells or 2-hour time point. (D) ST1926 elicits an early DNA damage response. Cells were treated with 1 μM ST1926 and immunoblotted against γ-H2AX antibodies at 2 hours after treatment. Similar trends in protein levels were observed in 2 independent experiments. (E) ST1926 reduces Tax protein levels in treated HuT-102 and MT-2 cells. Cells were treated with 1 μM ST1926 for up to 24 hours and immunoblotted against Tax antibodies. Similar trends in protein levels were observed in 3 independent experiments. All blots were reprobed with GAPDH antibody to ensure equal protein loading. (F) ST1926, but not CD437 or HPR, decreases Tax protein levels in treated MT-2 cells. Cells were treated with 1 μM ST1926 for up to 24 hours and immunoblotted against Tax antibodies. Similar trends in protein levels were observed in 2 independent experiments. All blots were reprobed with GAPDH antibody to ensure equal protein loading. (G) ST1926 reduces gag (p24, p19, and p15) protein levels in treated HuT-102 and MT-2 cells. Cells were treated with 1 μM ST1926 at the indicated time points and immunoblotted against sera derived from ATL patients.

ST1926 induces partially caspase-dependent apoptosis and results in elevated levels of p53 and γ-H2AX proteins and in reduced Tax protein levels. (A) ST1926 causes PARP and caspase 3 cleavage in HuT-102, MT-2, Jurkat, and MOLT-4 cells. Total sodium dodecyl sulfate protein lysates (50 µg/lane) were prepared after treating malignant T cells with 1 μM ST1926 for up to 48 hours. Lysates were immunoblotted against PARP and caspase-3 antibodies. Arrows indicate cleaved PARP and caspase 3. (B) Effects of the general caspase inhibitor z-VAD on ST1926-induced growth inhibition of HuT-102, MT-2, Jurkat, and MOLT-4 cells. Cells were pretreated for 2 hours with 100 μM z-VAD, followed by 24-hour treatment with 1 μM ST1926. Cell viability was determined in quadruplicate wells by the trypan blue dye exclusion and expressed as percentage of control. Results are an average of 2 independent experiments ± SE. *Statistically significant (P < .05) differences (independent t test). (C) ST1926 upregulates p53 and its phosphorylated form (P-p53) in malignant T cells. HuT-102, MT-2, Jurkat, and MOLT-4 cells were treated with 1 μM ST1926 for up to 48 hours and immunoblotted against p53 and P-p53 antibodies. Similar trends in protein levels were observed in 2 independent experiments. Bands were quantified, and results are expressed relative to control cells or 2-hour time point. (D) ST1926 elicits an early DNA damage response. Cells were treated with 1 μM ST1926 and immunoblotted against γ-H2AX antibodies at 2 hours after treatment. Similar trends in protein levels were observed in 2 independent experiments. (E) ST1926 reduces Tax protein levels in treated HuT-102 and MT-2 cells. Cells were treated with 1 μM ST1926 for up to 24 hours and immunoblotted against Tax antibodies. Similar trends in protein levels were observed in 3 independent experiments. All blots were reprobed with GAPDH antibody to ensure equal protein loading. (F) ST1926, but not CD437 or HPR, decreases Tax protein levels in treated MT-2 cells. Cells were treated with 1 μM ST1926 for up to 24 hours and immunoblotted against Tax antibodies. Similar trends in protein levels were observed in 2 independent experiments. All blots were reprobed with GAPDH antibody to ensure equal protein loading. (G) ST1926 reduces gag (p24, p19, and p15) protein levels in treated HuT-102 and MT-2 cells. Cells were treated with 1 μM ST1926 at the indicated time points and immunoblotted against sera derived from ATL patients.

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