Fig. 3. TAAR1 agonist RO5166017 increased Bcl-2 levels, but not Bax levels in HEK293 cells stably expressing mTAAR1. (A) RO5166017 dose dependently stimulated cAMP production in HEK-mTAAR1 cells. (B) Representative time course of RO5166017 treatment. Nontransfected or HEK-mTAAR1 cells were harvested 0, 2, 4, 6, and 18 hours after treatment with 100 nM RO5166017 (RO). Bcl-2, Bax, and the internal control, β-actin, were measured by western blot. (C) RO5166017 increased Bcl-2 levels in cells stably expressing mTAAR1 in a time-dependent manner; however, there was no agonist-induced regulation of Bcl-2 in nontransfected HEK293 cells. For Bcl-2, two-way ANOVA indicated an interaction between time × cell type [F(4,28) = 7.165, P < 0.001]. (D) Bax expression was not changed in nontransfected HEK293 or HEK-mTAAR1 cells. (E) The ratio of Bax/Bcl-2 was significantly lower in HEK-mTAAR1 cells than in nontransfected HEK293 cells. For the Bax/Bcl-2 ratio, there was a significant interaction between time × cell type [F(4,28) = 5.546, P < 0.01] by two-way ANOVA. Data shown represent the average of independent experiments, each time point conducted in duplicate. **P < 0.01; ***P < 0.001 vs. time (0 hour), +P < 0.05; +++P < 0.001 between two cell lines. Data are mean ± S.D.