Figure 5.
Figure 5. PIM inhibition downregulates NFκB and STAT3/5 activity in cHL-derived cell lines. (A) PIM1/2/3 depletion decreases STAT3/5 activity in RS cells. Cells were transfected with a nontargeting siRNA (control, Ctrl) or with a siRNA cocktail targeting all 3 PIM kinases (3×PIM). Thereafter, cells were cultured for 4 days, and phospho-STAT5 (Y694) and phospho-STAT3 (Y705) protein levels were assessed by immunoblotting. Densitometric quantifications of band intensities (p-STAT5 and p-STAT3 normalized to STAT5 and STAT3, respectively) are indicated above the lanes. (B) SEL24-B489 decreases STAT3/5 activity in RS cells. Cells were incubated for 24 hours with 3-5 µM SEL24-B489 or DMSO alone, harvested, and lysed. Phospho-STAT5 (Y694) phospho-STAT3 (Y705) levels were assessed by immunoblotting. Densitometric quantifications of band intensities are shown in supplemental Table 7. (C) PIM1/2/3 depletion decreases PIM-specific RELA/p65-S276 phosphorylation in RS cells. Cells were transfected with a nontargeting siRNA (control, Ctrl) or with a siRNA cocktail targeting all 3 PIM kinases (3×PIM). Thereafter, cells were cultured for 4 days, and phospho-RELA/p65 (S276) levels were assessed by immunoblotting. Densitometric quantifications of band intensities (p-RELA/p65 normalized to RELA/p65) are indicated above the lanes. (D) SEL24-B489 decreases RELA/p65 S276 phosphorylation in RS cells. Cells were incubated for 4 hours with DMSO alone or with 0.1-10 µM SEL24-B489, harvested, and lysed. The abundance of phospho-RELA/p65 (S276) was assessed by immunoblotting. GAPDH served as a loading control. Densitometric quantifications of band intensities are shown in supplemental Table 8. (E) SEL24-B489 reduces DNA binding activity of the RELA-containing NFκB complexes. RS cells were incubated either with DMSO alone or SEL24-B489 (5 µM) for 24 hours, lysed, and subjected to TransAM DNA binding assay, using the RELA-specific antibody. (F) PIM inhibition in cHL cell lines decreased NFκB-dependent gene expression. Cells were incubated with DMSO alone or with SEL24-B489 (3 µM) for 24 hours. Thereafter, expression of BCL2A1 and RELB was assessed by qPCR. Raw ΔCT values are shown in supplemental Figure 12. P values were determined using the 2-sided Gosset’s t-test: *P < .05; **P < .01; ***P < .001. Data in A-F are representative of 3 independent experiments.

PIM inhibition downregulates NFκB and STAT3/5 activity in cHL-derived cell lines. (A) PIM1/2/3 depletion decreases STAT3/5 activity in RS cells. Cells were transfected with a nontargeting siRNA (control, Ctrl) or with a siRNA cocktail targeting all 3 PIM kinases (3×PIM). Thereafter, cells were cultured for 4 days, and phospho-STAT5 (Y694) and phospho-STAT3 (Y705) protein levels were assessed by immunoblotting. Densitometric quantifications of band intensities (p-STAT5 and p-STAT3 normalized to STAT5 and STAT3, respectively) are indicated above the lanes. (B) SEL24-B489 decreases STAT3/5 activity in RS cells. Cells were incubated for 24 hours with 3-5 µM SEL24-B489 or DMSO alone, harvested, and lysed. Phospho-STAT5 (Y694) phospho-STAT3 (Y705) levels were assessed by immunoblotting. Densitometric quantifications of band intensities are shown in supplemental Table 7. (C) PIM1/2/3 depletion decreases PIM-specific RELA/p65-S276 phosphorylation in RS cells. Cells were transfected with a nontargeting siRNA (control, Ctrl) or with a siRNA cocktail targeting all 3 PIM kinases (3×PIM). Thereafter, cells were cultured for 4 days, and phospho-RELA/p65 (S276) levels were assessed by immunoblotting. Densitometric quantifications of band intensities (p-RELA/p65 normalized to RELA/p65) are indicated above the lanes. (D) SEL24-B489 decreases RELA/p65 S276 phosphorylation in RS cells. Cells were incubated for 4 hours with DMSO alone or with 0.1-10 µM SEL24-B489, harvested, and lysed. The abundance of phospho-RELA/p65 (S276) was assessed by immunoblotting. GAPDH served as a loading control. Densitometric quantifications of band intensities are shown in supplemental Table 8. (E) SEL24-B489 reduces DNA binding activity of the RELA-containing NFκB complexes. RS cells were incubated either with DMSO alone or SEL24-B489 (5 µM) for 24 hours, lysed, and subjected to TransAM DNA binding assay, using the RELA-specific antibody. (F) PIM inhibition in cHL cell lines decreased NFκB-dependent gene expression. Cells were incubated with DMSO alone or with SEL24-B489 (3 µM) for 24 hours. Thereafter, expression of BCL2A1 and RELB was assessed by qPCR. Raw ΔCT values are shown in supplemental Figure 12. P values were determined using the 2-sided Gosset’s t-test: *P < .05; **P < .01; ***P < .001. Data in A-F are representative of 3 independent experiments.

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