Figure 3
Figure 3. GCS-100 is associated with a time- and dose-dependent reduction of critical prosurvival proteins and up-regulation of the proapoptotic protein NOXA. (A) Whole-cell lysates were prepared from RPMI 8226 cells cultured with 500 μg/mL GCS-100 for up to 48 hours, and protein expression was examined by Western blot. A total of 25 μg of protein was separated by 12% SDS-PAGE. Protein was transferred to PVDF membrane and probed with the indicated antibodies to prosurvival BCL-2 family proteins. β-Actin was used to ensure equal loading. (B) RPMI 8226 or U266 cells were exposed to the indicated concentration of GCS-100 for 24 hours. Whole-cell lysates were prepared, and 50 μg of protein was separated by 12% SDS-PAGE, transferred to PVDF membrane, and probed with anti–MCL-1 antibody. β-Actin was used to demonstrate equal protein loading. (C) Whole-cell lysates were prepared from RPMI 8226 cells cultured with 500 μg/mL GCS-100 for up to 48 hours, and protein expression was examined by Western blot. A total of 25 μg of protein was separated by 12% or 15% SDS-PAGE. Protein was transferred to PVDF membrane and probed with the indicated antibodies. β-Actin was used to ensure equal loading. (D) RPMI 8226 cells were incubated with the pan-caspase inhibitor Z-VAD-fmk (50μM) or dimethyl sulfoxide (DMSO) for 1 hour and then were cultured in the presence or absence of GCS-100 (500 μg/mL) for 24 hours. Protein expression was examined by Western blot (25 μg of protein, 12% SDS-PAGE). indicates caspase cleavage products. β-Actin was used as a loading control. (E-G) The effect of caspase inhibition on GCS-100 mediated loss of mitochondrial transmembrane potential. RPMI 8226 cells were incubated for 1 hour with Z-VAD-fmk or DMSO and then cultured with GCS-100 (500 μg/mL) for 48 hours after which uptake of DiOC6(3) was assessed by flow cytometry as previously described. The proportion of DiOC6(3) low cells (E), DAPI-negative/DiOC6(3) low cells (F), and DAPI-positive cells (G) was assessed. GCS-100 on the x-axis indicates preincubation with DMSO (Z-VAD-fmk control), and Z-VAD-fmk on x-axis indicates preincubation with Z-VAD-fmk followed by GCS-100. Results are mean ± SEM of 3 independent experiments. *Significant difference (P ≤ .05) from untreated control. DiOC6(3).

GCS-100 is associated with a time- and dose-dependent reduction of critical prosurvival proteins and up-regulation of the proapoptotic protein NOXA. (A) Whole-cell lysates were prepared from RPMI 8226 cells cultured with 500 μg/mL GCS-100 for up to 48 hours, and protein expression was examined by Western blot. A total of 25 μg of protein was separated by 12% SDS-PAGE. Protein was transferred to PVDF membrane and probed with the indicated antibodies to prosurvival BCL-2 family proteins. β-Actin was used to ensure equal loading. (B) RPMI 8226 or U266 cells were exposed to the indicated concentration of GCS-100 for 24 hours. Whole-cell lysates were prepared, and 50 μg of protein was separated by 12% SDS-PAGE, transferred to PVDF membrane, and probed with anti–MCL-1 antibody. β-Actin was used to demonstrate equal protein loading. (C) Whole-cell lysates were prepared from RPMI 8226 cells cultured with 500 μg/mL GCS-100 for up to 48 hours, and protein expression was examined by Western blot. A total of 25 μg of protein was separated by 12% or 15% SDS-PAGE. Protein was transferred to PVDF membrane and probed with the indicated antibodies. β-Actin was used to ensure equal loading. (D) RPMI 8226 cells were incubated with the pan-caspase inhibitor Z-VAD-fmk (50μM) or dimethyl sulfoxide (DMSO) for 1 hour and then were cultured in the presence or absence of GCS-100 (500 μg/mL) for 24 hours. Protein expression was examined by Western blot (25 μg of protein, 12% SDS-PAGE). indicates caspase cleavage products. β-Actin was used as a loading control. (E-G) The effect of caspase inhibition on GCS-100 mediated loss of mitochondrial transmembrane potential. RPMI 8226 cells were incubated for 1 hour with Z-VAD-fmk or DMSO and then cultured with GCS-100 (500 μg/mL) for 48 hours after which uptake of DiOC6(3) was assessed by flow cytometry as previously described. The proportion of DiOC6(3) low cells (E), DAPI-negative/DiOC6(3) low cells (F), and DAPI-positive cells (G) was assessed. GCS-100 on the x-axis indicates preincubation with DMSO (Z-VAD-fmk control), and Z-VAD-fmk on x-axis indicates preincubation with Z-VAD-fmk followed by GCS-100. Results are mean ± SEM of 3 independent experiments. *Significant difference (P ≤ .05) from untreated control. DiOC6(3).

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