PIM expression in RS cells is regulated by NFκB and JAK-STAT activity. (A) Expression of PIM1, PIM2, and PIM3 after inhibition of the canonical NFκB signaling by expression of an IκBα super-repressor (SR-IκBα) in SUP-HD1 RS cells. Gene expression was assessed by qPCR and presented as relative changes using the 2^−ΔΔCT method. IκB, a direct target of canonical NFκB pathway, was used as a control. Raw ΔC_T (C_{T gene of interest} − C_{T GAPDH}) values are shown in supplemental Figure 2. P values were determined using 2-sided Gosset’s t-test. *P < .05; **P < .01; ***P < .001. (B) Expression of PIM1, PIM2, and PIM3 after siRNA-mediated RELA and RELB knock-down in SUP-HD1 cells. Cells were transfected with a nontargeting siRNAs pool (control, Ctrl) or siRNAs targeting RELA, RELB, or RELA and RELB (2×Rel). Cells were cultured for 4 days and assessed for changes in PIM1, PIM2, and PIM3 gene expression, using qPCR. Raw ΔC_T values are shown in supplemental Figure 5. P values were determined using 2-sided Gosset’s t-test: *P < .05; **P < .01; ***P < .001. (C) Expression of PIM1, PIM2, and PIM3 after JAK inhibition. Cell lines were incubated with 1-3 µM CYT387 or DMSO alone for 24 hours, harvested and analyzed by western blot, using indicated antibodies. GAPDH was used as a loading control. Densitometric quantifications of band intensities are shown in supplemental Table 5. Data in A-C are representative of 3 independent experiments.