Figure 7
Figure 7. IL-6 signaling induces IgM secretion. (A) BCWM.1 cells were serum-starved and treated with 50 ng/mL IL-6 for 15 minutes. After fixation and permeabilization, cells were probed for the phosphorylated forms of STATs 1-6. The blue histogram represents the isotype control; green represents baseline STAT phosphorylation in untreated BCWM.1 cells; red represents STAT phosphorylation on IL-6 stimulation. This experiment was performed 3 times, and representative histograms are shown. (B) HS-5 cells were plated in 6-well plates overnight and then serum starved overnight. Cells were then treated with 50 ng/mL IL-6 for the indicated times. Cells were then lysed and lysates used to determine activation of MAPK and JAK/STAT signaling pathways by immunoblotting. This experiment was repeated 3 times with similar results (C) IgM secretion by serum starved BCWM.1 cells cultured in the presence or absence of JAKI inhibitor (300nM) or DMSO control for 30 minutes and, then stimulated with IL-6 (50 ng/mL) for 3 days. To determine inhibition of STAT3 by JAKI inhibitor a Western blot was run with whole-cell lysates from serum starved BCWM.1 cultured in the presence of JAKI inhibitor (300nM) for 30 minutes and then treated with IL-6 (50 ng/mL) for 30 minutes. Immunoblots were repeated 2 times with similar results. (D) IgM secretion by serum starved BCWM.1 cells pretreated with or without the ERK inhibitor PD98059 (PD) or DMSO for 30 minutes and then treated with IL-6 (50 ng/mL) as indicated. Cell supernatants were harvested after 3 days and used to determine IgM secretion by ELISA. Experiments were performed twice in triplicate. Inhibition of Erk1/2 phosphorylation was confirmed by immunoblotting by culturing serum starved BCWM.1 in the presence or absence of PD inhibitor (50μM) for 30 minutes and then treated with IL-6 (50 ng/mL) for 30 minutes. Immunoblots were run twice with similar results. (E-F) Viability in the presence of either a pan-JAK (E) or MEK (F) inhibitor was assessed using annexin/propidium iodine staining. Experiments were performed 3 times. Data are presented as the average viability ± SEM.

IL-6 signaling induces IgM secretion. (A) BCWM.1 cells were serum-starved and treated with 50 ng/mL IL-6 for 15 minutes. After fixation and permeabilization, cells were probed for the phosphorylated forms of STATs 1-6. The blue histogram represents the isotype control; green represents baseline STAT phosphorylation in untreated BCWM.1 cells; red represents STAT phosphorylation on IL-6 stimulation. This experiment was performed 3 times, and representative histograms are shown. (B) HS-5 cells were plated in 6-well plates overnight and then serum starved overnight. Cells were then treated with 50 ng/mL IL-6 for the indicated times. Cells were then lysed and lysates used to determine activation of MAPK and JAK/STAT signaling pathways by immunoblotting. This experiment was repeated 3 times with similar results (C) IgM secretion by serum starved BCWM.1 cells cultured in the presence or absence of JAKI inhibitor (300nM) or DMSO control for 30 minutes and, then stimulated with IL-6 (50 ng/mL) for 3 days. To determine inhibition of STAT3 by JAKI inhibitor a Western blot was run with whole-cell lysates from serum starved BCWM.1 cultured in the presence of JAKI inhibitor (300nM) for 30 minutes and then treated with IL-6 (50 ng/mL) for 30 minutes. Immunoblots were repeated 2 times with similar results. (D) IgM secretion by serum starved BCWM.1 cells pretreated with or without the ERK inhibitor PD98059 (PD) or DMSO for 30 minutes and then treated with IL-6 (50 ng/mL) as indicated. Cell supernatants were harvested after 3 days and used to determine IgM secretion by ELISA. Experiments were performed twice in triplicate. Inhibition of Erk1/2 phosphorylation was confirmed by immunoblotting by culturing serum starved BCWM.1 in the presence or absence of PD inhibitor (50μM) for 30 minutes and then treated with IL-6 (50 ng/mL) for 30 minutes. Immunoblots were run twice with similar results. (E-F) Viability in the presence of either a pan-JAK (E) or MEK (F) inhibitor was assessed using annexin/propidium iodine staining. Experiments were performed 3 times. Data are presented as the average viability ± SEM.

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