Figure 3.

Downstream targets of Grm3 signaling

(A) Fluorescence resonance energy transfer (FRET)-based analysis of forskolin-induced cyclic adenosine monophosphate (cAMP) levels upon treatment with solvent (DMSO), Grm2/3 antagonist (RO1) at 100 nM, Grm2/3 agonist LY-379268 (LY) at 100 nM, or both (∗∗p < 0.01, ∗∗∗p < 0.001, t test). (B) Immunoblot of indicated proteins. ZH-161 cells were incubated in glutamate- and serum-free medium overnight prior to treatment for 5 min with RO1 or RO2 at 100 nM. (C) Clonogenic survival assay of indicated GSCs co-treated with RO1 or temozolomide (TMZ) or both; 100 cells per well were seeded in 96-well plates in six technical replicates and treated after 24 h. Metabolic activity was assessed utilizing the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay on days 7 (S24) or 21 (T-325; ∗∗p < 0.01, ∗∗∗p < 0.001, t test, referring to mono-treatment with RO1 at 100 nM versus DMSO solvent control; n.s., not significant, two-way ANOVA followed by Tukey’s post hoc test referring to concentration response of TMZ in RO1 versus DMSO). (D) Immunoblot of MGMT in T-325 cells treated with DMSO, RO1, and TMZ as indicated for 48 h.