Figure 6
Figure 6. Chemotherapy drugs reduce STAT4 protein levels via ubiquitin-mediated proteasomal degradation. NKL cells were treated without (-D) or with 50μM carmustine (Car) or 2μM etoposide (Eto) as described in Figure 3D. Dead cells were removed by Ficoll centrifugation from the culture of each treatment, and the collected viable cells were subsequently incubated with cycloheximide (CHX) for 0, 2, 4, 6, 8, 10, 12, and 24 hours. STAT4 protein expression was analyzed by immunoblotting, and a vertical line has been inserted to indicate the repositioned gel lane (A). The levels of STAT4 protein were determined by the densitometry of the corresponding bands normalized to endogenous control β-actin using the National Institutes of Health ImageJ program. The half-life of STAT4 protein was calculated accordingly, and the results shown are means ± SD from a total of 3 independent experiments (B). *P < .05 relative to treatment without chemotherapy drug (-D). (C) NKL cells were treated without (-D) or with 50μM carmustine (Car) or 2μM etoposide (Eto) as in Figure 3D. Dead cells were removed as described in panel A, followed by total protein extraction. Ubiquitin-conjugated protein was first enriched using the Ubiquitin Enrichment Kit (Thermo Scientific) with a specialized affinity resin binding to polyubiquitinylated proteins from cell lysates (Ub-beads). The negative control is the resin slurry lacking affinity to polyubiquitinylated proteins (beads). The enriched proteins were subjected to immunoblotting. Ubiquitin-conjugated STAT4 protein levels were analyzed using an anti-STAT4 mAb (left panel). Total STAT4 protein levels were analyzed using immunoblotting from 10 μg of whole-cell lysates (right panel). The ratio of Ub-STAT4 to total STAT4 is indicated below. The molecular weight (MW) at 75 and 118 kD is labeled. The panel is representative of 3 independent experiments, and a vertical line has been inserted to indicate the repositioned gel lane. (D) NKL cells were incubated with the proteasome inhibitor bortezomib at 5.2nM simultaneously with 50μM carmustine (Car) or 2μM etoposide (Eto) for 2 days. STAT4 protein levels were determined by immunoblotting. The results are representative of 3 independent experiments. Ratio of total STAT4 to β-actin is indicated below. (E) Densitometric analysis of STAT4 protein levels in NKL cells treated with carmustine or etoposide in the presence or absence of bortezomib. The results are presented as the averaged ratio of STAT4 to β-actin from 2 independent experiments as means ± SD (F) NKL cell treated as described in panel D were stimulated with IL-12 at 2 ng/mL for 1 day. The IFN-γ levels in the cell supernatants were evaluated using ELISA. Results are averaged from 2 independent experiments as means ± SD.

Chemotherapy drugs reduce STAT4 protein levels via ubiquitin-mediated proteasomal degradation. NKL cells were treated without (-D) or with 50μM carmustine (Car) or 2μM etoposide (Eto) as described in Figure 3D. Dead cells were removed by Ficoll centrifugation from the culture of each treatment, and the collected viable cells were subsequently incubated with cycloheximide (CHX) for 0, 2, 4, 6, 8, 10, 12, and 24 hours. STAT4 protein expression was analyzed by immunoblotting, and a vertical line has been inserted to indicate the repositioned gel lane (A). The levels of STAT4 protein were determined by the densitometry of the corresponding bands normalized to endogenous control β-actin using the National Institutes of Health ImageJ program. The half-life of STAT4 protein was calculated accordingly, and the results shown are means ± SD from a total of 3 independent experiments (B). *P < .05 relative to treatment without chemotherapy drug (-D). (C) NKL cells were treated without (-D) or with 50μM carmustine (Car) or 2μM etoposide (Eto) as in Figure 3D. Dead cells were removed as described in panel A, followed by total protein extraction. Ubiquitin-conjugated protein was first enriched using the Ubiquitin Enrichment Kit (Thermo Scientific) with a specialized affinity resin binding to polyubiquitinylated proteins from cell lysates (Ub-beads). The negative control is the resin slurry lacking affinity to polyubiquitinylated proteins (beads). The enriched proteins were subjected to immunoblotting. Ubiquitin-conjugated STAT4 protein levels were analyzed using an anti-STAT4 mAb (left panel). Total STAT4 protein levels were analyzed using immunoblotting from 10 μg of whole-cell lysates (right panel). The ratio of Ub-STAT4 to total STAT4 is indicated below. The molecular weight (MW) at 75 and 118 kD is labeled. The panel is representative of 3 independent experiments, and a vertical line has been inserted to indicate the repositioned gel lane. (D) NKL cells were incubated with the proteasome inhibitor bortezomib at 5.2nM simultaneously with 50μM carmustine (Car) or 2μM etoposide (Eto) for 2 days. STAT4 protein levels were determined by immunoblotting. The results are representative of 3 independent experiments. Ratio of total STAT4 to β-actin is indicated below. (E) Densitometric analysis of STAT4 protein levels in NKL cells treated with carmustine or etoposide in the presence or absence of bortezomib. The results are presented as the averaged ratio of STAT4 to β-actin from 2 independent experiments as means ± SD (F) NKL cell treated as described in panel D were stimulated with IL-12 at 2 ng/mL for 1 day. The IFN-γ levels in the cell supernatants were evaluated using ELISA. Results are averaged from 2 independent experiments as means ± SD.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal