Figure 4. 2AT-evoked increased phosphorylated ERK signal intensity is specific for F2rl1-expressing neurons.

Primary mouse DRG cultures were prepared for RNAscope in situ hybridization and immunocytochemistry. (A) Representative original magnification ×40 images of P2rx3 and F2rl1 mRNA and Neurofilament 200 (NF200) protein signal of cultured DRG neurons from WT mice. White arrows indicate a neuron positive for F2rl1 mRNA signal. Smaller image panels display zoomed-in images of the indicated single neuron. Scale bar: 20 μm. Zoomed-in image scale bar: 2 μm. (B) Pie chart illustrating the percentage distribution of neuronal F2rl1 and P2rx3 expression in vitro. About 3%–4% of primary cultured DRG neurons are F2rl1⁺, almost all of which are also P2rx3⁺. (C) Representative images of F2rl1 mRNA signal and phosphorylated ERK (p-ERK) immunolabeling in cultured DRG neurons from WT mice after treatment with vehicle or 2AT (1 μM) for 10 minutes. Scale bar: 2 μm. F2rl1⁺ DRG neurons treated with 2AT show increased p-ERK signal when compared with vehicle treatment. (D) Signal intensity of p-ERK increased markedly in F2rl1⁺ neurons after treatment with 2AT (1 μM) when compared with the vehicle treatment group. No significant difference in p-ERK signal intensity is seen between vehicle- and 2AT-treated groups in the F2rl1^– neurons. p-ERK signal was quantified through mean gray intensity value and normalized to the average p-ERK signal intensity value for the vehicle treatment groups. n = 15 and n = 16 for F2rl1⁺ neurons treated with vehicle or 2AT, respectively. n = 88 for both vehicle and 2AT treatment groups in F2rl1^– neurons. Data represent mean ± SEM. One-way ANOVA with Bonferroni’s multiple comparisons (D) ***P < 0.001.