Fig 1: PACAP38-induced proliferation of SH-SY5Y cells is mediated via the Gαs/EPAC/AKT-ERK signaling pathway. A SH-SY5Y cell proliferation was measured using the CCK-8 assay. Cells were pretreated for 30 min with vehicle (0.001% DMSO, v/v) or the following inhibitors: Gαq/11 inhibitor YM-254,890 (100 nM), AC inhibitor KH7 (10 µM), PKA inhibitor PKI-(6–22)-amide (10 µM), EPAC inhibitor ESI-09 (10 µM), or RhoA inhibitor rhosin (10 µM), followed by treatment with PACAP38 (10 nM). Statistical analysis was performed using unpaired two-tailed Student’s t-test. **P < 0.01; ns, not significant. B Validation of Gαs knockout in SH-SY5Y cells. Cells were transduced with lentiviruses encoding sgControl or sgRNAs targeting GNAS (sgGNAS #1 and sgGNAS #2). Gαs protein levels were assessed by western blotting following puromycin selection. C Effects of Gαs knockout on PACAP38-induced proliferation. Control and Gαs-deficient SH-SY5Y cells were stimulated with increasing concentrations of PACAP38 for 72 h, and proliferation was measured using the CCK-8 assay. D-F SH-SY5Y cells were pretreated for 30 min with MEK inhibitor U0126 (D), PI3K inhibitor LY294002 (E), or AKT inhibitor AKTi-1/2 (F), followed by PACAP38 (10 nM) treatment. Cell proliferation was assessed using the CCK-8 assay. Statistical analysis was performed using unpaired two-tailed Student’s t-test. *P < 0.05 and **P < 0.01 for comparisons between vehicle and inhibitor-treated groups, and #P < 0.05, ##P < 0.01, and ###P < 0.001 for comparisons between PACAP38 alone and PACAP38 plus inhibitor-treated groups (marked in blue). ns, not significant. G Effect of Gαs knockout on PACAP38-induced ERK and AKT phosphorylation. Control and Gαs-deficient SH-SY5Y cells were treated with PACAP38 (10 nM) for the indicated time points, and ERK and AKT activation was evaluated by western blot analysis. Statistical analysis was performed using unpaired two-tailed Student’s t-test. *P < 0.05. Data represent the mean ± SEM of n = 3 independent experiments