WSS stimulates cAMP–PKA signaling and phosphorylation of CREB. (A) Expression of CREB, cAMP–PKA, and calmodulin signaling components believed to lie downstream of noncanonical Wnt/calcium signaling are altered by WSS and inhibition of PGE₂ by indomethacin at 6 and 36 h (n = 3 independent experiments at each time point). (B) Phosphorylation of ser133 on CREB (P-CREB) is elevated by 3 h of WSS. (C) P-CREB is reduced in Ncx1^−/− PSp (n = 6 mutant, n = 8 wild-type littermates analyzed). Representative data from different gels are separated by white space. (D) Intracellular and circulating (secreted) cAMP is increased by WSS in a PGE₂-dependent manner (n = 3 independent experiments; two-way ANOVA: **, P < 0.001). Indomethacin significantly reduced levels of intracellular cAMP (Holm-Šídák comparison: P = 0.009) and circulating cAMP (P = 0.005). Stimulation of cAMP synthesis by adenylyl cyclase with forskolin serves as a positive control. (E) Blocking antibodies against PGE₂ or inhibitors of COX1/2 effectively suppress WSS-induced progenitor activity in M3434 methylcellulose assays. PKA inhibition by H89 similarly reduces progenitor activity, whereas stimulation of cAMP levels by forskolin elevates hematopoietic activity above static vehicle controls (n = 3 independent experiments; two-way ANOVA: **, P < 0.001). (F) Interruption of prostaglandin signaling by indomethacin reduces transcription of Ras/MAPK/Akt, CREB, and calcineurin/NFAT pathway components related to calcium transport and calmodulin kinase activity (p-value cutoff 0.05, FDR 0.8, overlap coefficient cutoff 0.5). Data are represented as mean ± SEM.