Inhibition of PRMT5 results in enhanced cell death and reduced growth of canine lymphoma cell lines and primary samples.

(A) Representative flow cytometry plots of cell line 17–71 treated with the PRMT5 inhibitor (C220) for 5 days demonstrating staining for early (Annexin-V+/PI-) and late apoptotic (Annexin-V+/PI+) cells. Cell viability was quantified by the percentage of Annexin-V/PI double negative staining. The percentage of viable, non-apoptotic cells is indicated in the lower left quadrant. (B) Canine lymphoma cell lines were treated with log10 increasing concentrations of C220 and cell viability was measured at day 3, 4, and 5. The beta mixed regression model supported significantly decreasing linear trends between viability and day all cell lines (17–71: *p < 0.04 for each concentration, CLBL-1: ***p < 0.001 for concentrations ≥ 0.1 μM, and OSW: ***p < 0.001 for concentrations ≥ 1 μM). (C) Canine lymphoma cell lines were treated with log10 increasing concentrations of C220 and viable cell proliferation was measured at day 3, 4, 5, and 6 by the MTS colorimetric assay for metabolic activity. For 17–71, the linear mixed model supported a significantly decreasing linear trend between MTS absorbance and day (17–71: *p < 0.012 for concentrations ≥ 0.1 μM). For cell lines CLBL-1 and OSW the main effect of both log10 treatment concentration and day are both estimated to decrease MTS absorbance by 0.12 units (CLBL-1: p < 0.001) and 0.09 (OSW: p < 0.001) for every one unit increase. (D) Induction of apoptosis in primary lymphoma samples treated ex-vivo (n = 3 dogs per condition) indicated by a statistically significant decrease in viability compared to the normalized vehicle control (*p < 0.05, two-tailed Student’s t-test). 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 respective p-values are indicated (*p < 0.05, **p < 0.01, ***p < 0.001).