Selectivity of PRMT5 inhibition with C220.

(A) PRMT5 inhibition is selectively toxic to canine lymphoma cells and not resting or activated ND B-cells or T-cells as determined by percent of viable cells over a 96-hour incubation. There are no statistically significant differences in predicted viabilities between any pairwise comparison of treatment concentration within the ND resting and activated B-cells or T-cells; whereas, the viability of the lymphoma cell lines (CLBL-1 and OSW) significantly decreased as treatment concentration increased (***p < 0.001 for each pairwise comparison to vehicle control). (B) Cell lines 17–71 (day 4) and CLBL-1 (day 2) and OSW (day 3) were treated at the indicated concentration of C220 (μM). Whole cell lysates were immunoblotted for the proteins indicated. Western blots were detected using Licor IRDye conjugated secondary antibodies and the Licor Odyssey imaging system. For all cell lines, effects of PRMT5 inhibition with C220 treatment on biomarkers of PRMT5 activity shows a dose-dependent decrease in global marks of symmetric di-methylarginine (SDMA) and symmetric di-methylation of histone H4 arginine-3 (H4R3me2s) with a minimal effect to PRMT5 or global asymmetric di-methylarginine (ADMA). (C) Immunoblots and quantification of three biological replicate experiments, showing attenuated SDMA and H4R3me2s in 17–71 (day 4) and CLBL-1 (day 2) after treatment with 100 nM C220, with no significant decreased to PRMT5 expression. PRMT5, SDMA, and H4R3me2s with B-actin were ran on separate gels. Immunoblots were detected using film and HRP conjugated secondary antibodies. Protein expression was quantified by the ratio of target protein to loading control then normalized to the 0 nM DMSO control. Statistical significance evaluated with a paired two-tailed Student’s t-test compared to normalized vehicle control. For all graphs data are shown relative to the 0 nM DMSO control and are the averages of three or more biological replicate experiments. Error bars show mean ± SEM and p-values are indicated as such (*p < 0.05, *p < 0.01, ***p < 0.001).