FIGURE 5 from Inhibition of NEK2 Promotes Chemosensitivity and Reduces KSHV-positive Primary Effusion Lymphoma Burden

NEK2 inhibition in PEL decreases expression and activity of the ABC transporter proteins, MDR1 and MRP. A, Western blot analysis for MDR1, MRP1, and BCRP in PEL cells treated with or without JH295. GAPDH was used as the loading control. Data are representative of three independent biological replicates. B, Western blot analysis for MDR1, MRP1, and BCRP in BC1 cells with intact NEK2 expression (NTC) or depleted NEK2 expression (shRNA). The accompanying Western blot analysis demonstrates depleted NEK2 expression in the BC1 cells used. GAPDH was used as the loading control. All data are representative of three independent biological replicates. C, Multidrug resistance assay measuring the activity of ABC transporter proteins in PEL cells treated with or without JH295. Data represent the percentage of fluorescent dye retained inside the cells after the efflux period. The 4°C sample represents a positive control (no activity; near 100% dye retention within the cells). Data are plotted as individual data points of three independent biological replicates representing mean ± SEM and analyzed using one-way ANOVA with Dunnett multiple comparisons. ***, P = 0.0003; **, P < 0.004; *, P = 0.02. D, Data are from the same experiments as C but used to calculate the multidrug resistance activity factor (MAF) for each treatment condition. Data are plotted as individual data points of three independent biological replicates representing mean ± SEM and analyzed using one-way ANOVA with Dunnett multiple comparisons. BCBL1 48 hours ***, P < 0.001; **, P = 0.0069; JSC1 48 hours ****, P < 0.0001; ***, P = 0.0003. E–G, eFLUXX-ID Green assay measuring the contribution of each individual ABC transporter protein to efflux activity in PEL cells treated with or without JH295 for 48 hours. Data are fold change in mean fluorescence intensity (MFI) relative to the no inhibitor control. Data are plotted as individual data points of three independent biological replicates representing mean ± SD and analyzed using two-way ANOVA with Dunnett multiple comparisons. BCBL1 **, P < 0.002; BC1 **, P < 0.009, *, P = 0.0352; JSC1 ***, P = 0.0003, **, P < 0.008. Corresponding histogram plots of FITC (green fluorescent dye) intensity within the PEL cells are gated on PI-negative single cells, normalized to mode, and are representative of three independent biological replicates.