Figure 3
Figure 3. IL-15 has no effect on apoptosis in SD-1 cells. (A) Intracellular IL-15 protein levels in SD-1 and Sup-B15 cells, measured by flow cytometry with isotype- (shaded) and IL-15–specific antibodies (open). Corrected mean fluorescence intensity (MFI) represents MFI IL-15PE − MFI isotype control. (B) Levels of IL-15Rα, β, and γ in SD-1 and Sup-B15 cells were determined by western blot with β-tubulin as a loading control. MOLT-4 cells expressing high levels of IL-15Rα were used as a positive control. (C) Western blot for BCL-xL and BCL-2 protein expression in SD-1 cells treated with IL-15 (1, 5, and 25 ng/mL) for 72 hours. (D) PARP cleavage in SD-1 cells treated with IL-15 (25 ng/mL) for 96 hours. The positive-control lane contains SD-1 cells treated with the apoptosis-inducing agent AA2 (50 μM) for 1 hour. Histone H3 was used as loading control. (E) Annexin V-FITC and propidium iodide staining of SD-1 cells treated with (right panel) or without (central panel) 25 ng/mL IL-15 for 96 hours. The positive control shows cells exposed to AA2 (50 μM) for 1 hour (left). (F) SD-1 cells (dark bars) and the dexamethasone sensitive cell line CCRF-CEM (light bars; used as a positive control) were exposed to vehicle (0.1% EtOH) or dexamethasone (100 nM, 1 μM, or 10 μM) for 48 hours. Percentage apoptosis (sum of early and late apoptosis) of these cells was then measured using an Annexin V assay. Data represent mean ± standard error of the mean with n = 3 for each cell line. (G) SD-1 (dark bars) and CCRF-CEM cells (light bars) were exposed to similar conditions as panel C for 48 hours, and viability was assessed using an MTT assay. Data show viability (% of control) compared with untreated control. (H) MTT assay of SD-1 (dark bars) and CCRF-CEM (light bars) cells exposed to dexamethasone (Dex; 10 µM) ± IL-15Rα nAb/isotype control for 72 hours. In panels F to H, data represent mean ± standard error of the mean (n = 3) and were analyzed using a Kruskal Wallis test with Dunn’s multiple comparison test. **P < .01, ***P < .001. FCS, fetal calf serum; PE, phycoerythrin; PI, propidium iodide.

IL-15 has no effect on apoptosis in SD-1 cells. (A) Intracellular IL-15 protein levels in SD-1 and Sup-B15 cells, measured by flow cytometry with isotype- (shaded) and IL-15–specific antibodies (open). Corrected mean fluorescence intensity (MFI) represents MFI IL-15PE − MFI isotype control. (B) Levels of IL-15Rα, β, and γ in SD-1 and Sup-B15 cells were determined by western blot with β-tubulin as a loading control. MOLT-4 cells expressing high levels of IL-15Rα were used as a positive control. (C) Western blot for BCL-xL and BCL-2 protein expression in SD-1 cells treated with IL-15 (1, 5, and 25 ng/mL) for 72 hours. (D) PARP cleavage in SD-1 cells treated with IL-15 (25 ng/mL) for 96 hours. The positive-control lane contains SD-1 cells treated with the apoptosis-inducing agent AA2 (50 μM) for 1 hour. Histone H3 was used as loading control. (E) Annexin V-FITC and propidium iodide staining of SD-1 cells treated with (right panel) or without (central panel) 25 ng/mL IL-15 for 96 hours. The positive control shows cells exposed to AA2 (50 μM) for 1 hour (left). (F) SD-1 cells (dark bars) and the dexamethasone sensitive cell line CCRF-CEM (light bars; used as a positive control) were exposed to vehicle (0.1% EtOH) or dexamethasone (100 nM, 1 μM, or 10 μM) for 48 hours. Percentage apoptosis (sum of early and late apoptosis) of these cells was then measured using an Annexin V assay. Data represent mean ± standard error of the mean with n = 3 for each cell line. (G) SD-1 (dark bars) and CCRF-CEM cells (light bars) were exposed to similar conditions as panel C for 48 hours, and viability was assessed using an MTT assay. Data show viability (% of control) compared with untreated control. (H) MTT assay of SD-1 (dark bars) and CCRF-CEM (light bars) cells exposed to dexamethasone (Dex; 10 µM) ± IL-15Rα nAb/isotype control for 72 hours. In panels F to H, data represent mean ± standard error of the mean (n = 3) and were analyzed using a Kruskal Wallis test with Dunn’s multiple comparison test. **P < .01, ***P < .001. FCS, fetal calf serum; PE, phycoerythrin; PI, propidium iodide.

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