Figure 5.
Figure 5. Reduction of IFN-γ protein by 15d-PGJ2 is mediated through MG132-sensitive and chloroquine-sensitive pathways. (A-C) Detection of intracellular IFN-γ protein by flow cytometry in permeabilized NK92 cells. NK92 cells were pretreated with 15d-PGJ2 (3 μM) or drug vehicle (no stimulation). IL-2 (200 U/mL) was then added for 4 hours. Detailed procedures are described in “Materials and methods.” The histogram plots are defined as follows: PE-anti-IFN-γ (solid line), PE-conjugated control antibody (dashed line), and the net difference between PE-anti-IFN-γ and PE-conjugated control antibody (filled). (D) Detection of extracellular IFN-γ protein in the supernatant by ELISA. Supernatant was collected from the same cells that were analyzed for detection of intracellular IFN-γ protein by flow cytometry and assayed for IFN-γ protein as described in “Materials and methods.” (E) Effect of MG132 or chloroquine on IFN-γ inhibition by 15d-PGJ2. NK92 cells were preincubated with 15d-PGJ2 (2 μM) for 1 hour, and then IL-2 (200 U/mL), MG132 (5 μM), lactacystin (5 μM), or chloroquine (1 μM) was added for 8 hours. The supernatant was collected at each point, and IFN-γ protein was measured by ELISA. Relative level of IFN-γ was presented as changes of percentage compared with IL-2-treated sample (100%). (F) Kinetic analysis of IFN-γ protein degradation. NK92 cells were treated with IL-2 (200 U/mL) for 3 hours, and then cycloheximide (5 μg/mL) was added to inhibit protein synthesis. The mixtures were divided into 4 aliquots: control, 15d-PGJ2 (2 μM), MG132 (5 μM), and MG132/15d-PGJ2. The time of cycloheximide addition was designated as the zero-hour point, and the amount of IFN-γ measured by ELISA at this time point was arbitrarily set as 100%. Remaining IFN-γ at each time point (%) = (amount of IFN-γ at this time point - amount of IFN-γ at the previous time point)/amount of IFN-γ at the previous time point × 100%. The equation for the curve is Y = span × e (K × X) + plateau, where span and plateau are fixed at 100% and 0%, respectively, and half-life = 0.69/K. Data represent the average from 3 different experiments (mean ± SE). *Statistically significant changes compared with control samples (P < .05).

Reduction of IFN-γ protein by 15d-PGJ2 is mediated through MG132-sensitive and chloroquine-sensitive pathways. (A-C) Detection of intracellular IFN-γ protein by flow cytometry in permeabilized NK92 cells. NK92 cells were pretreated with 15d-PGJ2 (3 μM) or drug vehicle (no stimulation). IL-2 (200 U/mL) was then added for 4 hours. Detailed procedures are described in “Materials and methods.” The histogram plots are defined as follows: PE-anti-IFN-γ (solid line), PE-conjugated control antibody (dashed line), and the net difference between PE-anti-IFN-γ and PE-conjugated control antibody (filled). (D) Detection of extracellular IFN-γ protein in the supernatant by ELISA. Supernatant was collected from the same cells that were analyzed for detection of intracellular IFN-γ protein by flow cytometry and assayed for IFN-γ protein as described in “Materials and methods.” (E) Effect of MG132 or chloroquine on IFN-γ inhibition by 15d-PGJ2. NK92 cells were preincubated with 15d-PGJ2 (2 μM) for 1 hour, and then IL-2 (200 U/mL), MG132 (5 μM), lactacystin (5 μM), or chloroquine (1 μM) was added for 8 hours. The supernatant was collected at each point, and IFN-γ protein was measured by ELISA. Relative level of IFN-γ was presented as changes of percentage compared with IL-2-treated sample (100%). (F) Kinetic analysis of IFN-γ protein degradation. NK92 cells were treated with IL-2 (200 U/mL) for 3 hours, and then cycloheximide (5 μg/mL) was added to inhibit protein synthesis. The mixtures were divided into 4 aliquots: control, 15d-PGJ2 (2 μM), MG132 (5 μM), and MG132/15d-PGJ2. The time of cycloheximide addition was designated as the zero-hour point, and the amount of IFN-γ measured by ELISA at this time point was arbitrarily set as 100%. Remaining IFN-γ at each time point (%) = (amount of IFN-γ at this time point - amount of IFN-γ at the previous time point)/amount of IFN-γ at the previous time point × 100%. The equation for the curve is Y = span × e (K × X) + plateau, where span and plateau are fixed at 100% and 0%, respectively, and half-life = 0.69/K. Data represent the average from 3 different experiments (mean ± SE). *Statistically significant changes compared with control samples (P < .05).

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