Figure 1.

Type I PRMT inhibitors abrogate toxicity associated with GR₁₅ and PR₁₅ challenge in NSC-34 cells. (A) Relative signal of total ADMe in NSC-34 cells after having been incubated with Type I PRMT inhibitors for 24 hours. Quantification of the signal was done by ICW assay using an antibody against total ADMe and normalized using an antibody against total protein (MS023: df = 132, R² = 0.7441; MS049: df = 82, R² = 0.8218; EPZ020411: df = 83, R² = 0.7087; GSK3368715: df = 50, R² = 50). (B) Schematic of an ICW assay workflow and example visualization. We used a primary antibody against total ADMe, and a fluorescent green IRDye secondary antibody. A red, CellTag700 antibody was used to fluorescently label total protein. (C, D) Percent metabolic activity after challenging with 3 µM of GR₁₅ or PR₁₅ and dosing with a Type I PRMT inhibitor (GR₁₅ challenge: MS023: df = 67, R² = 0.8054; MS049: df = 10, R² = 0.9630; EPZ020411: df = 13, R² = 0.9237; GSK3368715: df = 19, R² = 0.8077; PR₁₅ challenge: MS023: df = 40, R² = 0.9034; MS049: df = 10, R² = 0.9571; EPZ020411: df = 13, R² = 0.9625; GSK3368715: df = 7, R² = 0.9330). (E, F) Percent LDH release after challenging with 3 µM of GR₁₅ or PR₁₅ and dosing with a Type I PRMT inhibitor (GR₁₅ challenge: MS023: df = 22, R² = 0.9056; MS049: df = 10, R² = 0.9414; EPZ020411: df = 13, R² = 0.8807; GSK3368715: df = 10, R² = 0.9313; PR₁₅ challenge: MS023: df = 22, R² = 0.9398; MS049: df = 10, R² = 0.9613; EPZ020411: df = 10, R² = 0.7078; GSK3368715: df = 10, R² = 0.9375). (G) Percent metabolic activity after challenging NSC-34 cells with 3 µM of GR₁₅ or PR₁₅ and dosing with GSK591 (two-way ANOVA with Dunnett’s multiple comparison; n = 6 untreated, n = 3 treated; P values > 0.1657, mean ± s.e.m). The 200 µM dose of GSK591 significantly decreased metabolic activity beyond the effect observed with 3 µM of GR₁₅ alone (two-way ANOVA with Sidak’s multiple comparison; n = 6 untreated, n = 3 treated; *P = 0.0261, mean ± s.e.m.) (H) Percent metabolic activity after challenging cells with 3 µM of GR₁₅ or PR₁₅ and dosing with MS094 (two-way ANOVA with Dunnett’s multiple comparison; NS P values >0.5496, mean ± s.e.m.). (I) Percent LDH release after challenging cells with 3 µM of GR₁₅ or PR₁₅ and dosing with MS094 (two-way ANOVA with Dunnett’s multiple comparison; NS P values >0.1650, mean ± s.e.m.). For (C, D, G, H), 100% activity represents untreated NSC-34 cells, and 0% activity represents metabolic activity after 3 µM of GR₁₅ or PR₁₅ challenge alone. For (C–F), full dose response plots can be found in Supplementary Figure 1 . For (A), (C–F), a full listing of n for each condition can be found in the Supplementary Statistics section of the Supplementary Material .