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. 2020 Sep 8;11:569661. doi: 10.3389/fphar.2020.569661

Figure 2.

Figure 2

Asymmetrical arginine dimethylation of GR15 prevents abrogation of toxicity by MS023. (A) Products of in-vitro methylation assay immunoblotted for asymmetrically dimethylated arginine 3 in Histone 4 and GR dipeptide (left) and total asymmetrical arginine dimethylation and GR dipeptide (right). Also shown is the peptide sequence of Histone 4 with the epitope of the H4R3me2a antibody highlighted. Both blots show ADMe of GR15 and that it is increasingly dimethylated with increasing amounts of PRMT1. (B) Percent metabolic activity after challenging with ADMe-GR15 compared to unmethylated GR15 (****P < 0.0001, ***P = 0.0002, **P = 0.0013, *P = 0.0281) or ADMe-PR15 compared to unmethylated PR15 (####P < 0.0001, ##P = 0.004; two-way ANOVA with Sidak’s multiple comparison; n = 3 for each dose of DRP; mean ± s.e.m.). (C) Percent LDH release after challenging with ADMe-GR15 compared to unmethylated GR15 (**P = 0.0083, *P = 0.0117) or ADMe-PR15 compared to PR15 (#P = 0.0239; two-way ANOVA with Sidak’s multiple comparison; n = 3 for each dose of DRP; NS P > 0.05 mean ± s.e.m.). (D) Percent metabolic activity after challenge with 3 µM of ADMe-GR15 or unmethylated GR15 and dosing with MS023, compared to activity after challenge of 3 µM of GR15 alone (two-way ANOVA with Dunnett’s multiple comparison; n = 9 for each dosing group; NS P > 1.638, ****P < 0.0001, *P = 0.0411; mean ± s.e.m.). (E) Percent LDH release after challenge with 3 µM of ADMe-GR15 or unmethylated GR15 and dosing with MS023, compared to release after challenge of 3 µM of GR15 alone (two-way ANOVA with Dunnett’s multiple comparison; n = 3 for each dosing group; NS, P > 0.0657, ****P < 0.0001, ***P = 0.0002; mean ± s.e.m.). (F) Percent metabolic activity after challenge with 3 µM of ADMe-PR15 or unmethylated PR15 and dosing with MS023, compared to activity after challenge of 3 µM of PR15 alone (two-way ANOVA with Dunnett’s multiple comparison; n = 3 for each dosing group; NS, P > 0.1830, ****P < 0.0001, ***P = 0.0004, #P > 0.0242; mean ± s.e.m.). (G) Percent LDH release after challenge with 3 µM of ADMe-PR15 or unmethylated PR15 and dosing with MS023, compared to release after challenge of 3 µM of PR15 alone (two-way ANOVA with Dunnett’s multiple comparison; n = 3 for each dosing group; NS, P > 0.0.2947, ****P < 0.0001, ***P = 0.0007; ####P < 0.0001, ###P = 0.0004, #P > 0.0273 mean ± s.e.m.). For (D, F), 100% activity represents untreated NSC-34 cells, and 0% activity represents metabolic activity after 3 µM of GR15 or PR15 challenge alone, respectively. For (F, G), *- PR15 + MS023 compared to PR15 alone. # - ADMe-PR15 + MS023 compared to PR15 alone.