Fig. 10

TDZ modulation of the ERK1/2 pathway. a, b Astrocytes were treated with medium alone (basal), or with the indicated concentrations of TDZ, or FLUOX (10 μM) or 5-HT (10 μM)for 30 min (a) or 24 h (b). Following incubation, the levels of phosphorylated and total ERK1/2 were evaluated using an ELISA kit as described in the Methods section. The data are expressed as phosphorylated/total ERK1/2 ratio. The data are the mean ± SEM of three independent experiments performed in triplicate. c Cells were pre-incubated with medium alone (basal), or 15 nM (S)-WAY 100135 (5-HT_1AR antagonist), or 30 nM (R)-DOI (5-HT_2A/CR agonist), or 250 nM clonidine (α-adrenergic receptor agonist), or 100 μM histamine (H₁ histamine receptor agonist). After 15 min, cells were incubated with TDZ (10 μM) for an additional 30 min. d Human astrocytes were pre-treated with medium alone (basal), or 15 nM (S)-WAY 100135 (5-HT_1AR antagonist), or 10 nM GR 127935 (5-HT_1B/DR antagonist), or 5 nM RS 127445 (5-HT_2BR antagonist), or 30 nM (R)-DOI (5-HT_2A/CR agonist) or 100 nM SR 57227 (5-HT₃R agonist). After 15 min, cells were incubated with FLUOX (10 μM) for an additional 30 min. Following incubation, the levels of phosphorylated and total ERK1/2 were evaluated using an ELISA kit. e Human astrocytes were pre-treated with 200 ng/ml PTX (G_αi/o inhibitor), 1 μM H89 (PKA inhibitor), or 1 μM bisindolylmaleimide (PKC inhibitor), or 500 nM wortmannin (PI3K inhibitor); then, cells were incubated with TDZ (10 μM) or FLUOX (10 μM) for an additional 30 min. Following incubation, the levels of phosphorylated and total ERK1/2 were evaluated using an ELISA kit. The significance of the differences was determined using a one-way ANOVA-Tukey HSD post hoc test: *P < 0.05, **P < 0.01 ***P < 0.01 vs. basal; ^## P < 0.01, ^### P < 0.001 vs cells stimulated with TDZ or FLUOX