PIM1, PIM2, and PIM3 kinases support RS cell survival. (A) SiRNA-mediated PIM1, PIM2, and PIM3 silencing in HDLM-2 cell line. Cells were transfected with a nontargeting siRNA (control, Ctrl), siRNAs targeting individual PIM isoforms, or with a siRNA cocktail targeting all 3 PIM kinases (3×PIM) and cultured for 4 days. Relative changes in transcript abundance are calculated using the 2−ΔΔCT method. Raw ΔCT values are shown in supplemental Figure 7. P values were determined using 2-sided Gosset’s t-test: *P < .05; **P < .01; ***P < .001). (B) SiRNA-mediated PIM1, PIM2, and PIM3 protein knock-down in HDLM-2 and SUP-HD1 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 PIM1, PIM2, and PIM3 expression was assessed by immunoblotting. Densitometric quantifications of band intensities (normalized to GAPDH) are indicated above the lanes. (C) Induction of apoptosis in HDLM-2 and SUP-HD1 RS cells after silencing PIM1, PIM2, and PIM3 expression individually or in combination. Cells were transduced with siRNAs as in A. Apoptosis was assessed by AnnexinV/7AAD staining. Bar graphs represent mean values derived from 3 independent experiments. P values were determined using 2-sided Gosset’s t-test: *P < .05. (D) EC50 dose-response curves for SEL24-B489 in cHL-derived cell lines. Cellular proliferation was determined by 3-(4,5 dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl)-2H-tetrazolium reduction assay after 72 hours of incubation. The individual data points on dose-response curves represent the mean ± SD. Calculated EC50 values are given below the plots. (E) Cellular apoptosis in SEL24-B489-treated RS cells. After 96 hours of incubation with 5 µM SEL24-B489, apoptosis was measured by AnnexinV/7AAD staining, followed by FACS analysis (summary and statistics of 3 independent experiments is shown in supplemental Figure 9). Data in A-E are representative of 3 independent experiments
