PIM inhibition attenuates expression of NFκB-regulated cytokines, chemokines, and immunoregulatory molecules PD-L1/2 and Gal-1 in RS cells. (A) 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 TNF, IL-8, CCL5, IL-13, and CD40 was assessed by qPCR. Raw ΔC_T values are shown in supplemental Figure 13. P values were determined using the 2-sided Gosset’s t-test: *P < .05; **P < .01; ***P < .001. (B) PIM depletion decreases Gal-1 expression 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 Gal-1 protein abundance was assessed by immunoblotting. GAPDH served as a loading control. Densitometric quantifications of band intensities (normalized to GAPDH) are indicated above the lanes. (C) Inhibition of PIM kinases attenuates Gal-1 protein expression in RS cells. CHL cells were incubated for 24 hours with 3-5 µM SEL24-B489 or DMSO alone, harvested, and lysed. Expression of Gal-1 was assessed by immunoblotting. GAPDH served as a loading control. Densitometric quantifications of band intensities (normalized to GAPDH) are indicated above the lanes. (D) PIM knock-down decreases surface PD-L1 and PD-L2 expression. HDLM-2 and SUP-HD1 RS 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 5 days, and cell surface expression of PD-L1 and PD-L2 was assessed by flow cytometry. (E) PIM inhibition decreases surface PD-L1 and PD-L2 expression. Cells were incubated with DMSO alone or with SEL24-B489 (2 µM) for 120 hours. Thereafter, cell surface expression of PD-L1 and PD-L2 was assessed by flow cytometry. Data in A-E are representative of 3 independent experiments.