Fig. 5: 8GL represses mTORC1 activity via the GPR132-PKA pathway in AML.

A Schematic illustration of the GPCR-Gs-PKA signaling axis that inhibits mTORC1. B HL60 cells were treated with 8GL (30 μM) for indicated times. The levels of p-S6K1 (Thr389) and p-CREB (Ser133) were measured using western blotting. C HL60 cells were treated with 8GL at concentrations of 0, 15, 30, and 60 μM. The levels of p-S6K1 (Thr389) and p-CREB (Ser133) were measured using western blotting. The antibodies were purchased from Cell Signaling Technology (CST). p-S6K1, CST-9205; S6K1, CST-9202; p-CREB, CST-9198; CREB, CST-9197. D Western blotting analysis of p-S6K1 (Thr389) and p-CREB (Ser133) in 8GL-treated GPR132 WT and GPR132 KO AML cell lines. 8GL treatment: 30 μM for 3 h. Sg1 and sg2 represent two distinct sgRNAs for GPR132. E Phosphorylation status of S6K1 (Thr389) and CREB (Ser133) in HL60 and MV4-11 cells incubated with 8GL (30 μM or 60 μM) and/or H89 (1 μM). F HL60 and MV4-11 cells were treated with 8GL (0 μM, 30 μM, and 60 μM) for 48 h, and c-Myc expression was detected using western blotting. G Western blot analysis of c-Myc protein in GPR132 WT and GPR132 KO AML cells in the presence of 8GL (30 μM for 48 h). Sg1 and sg2 represent two distinct sgRNAs for GPR132. H Gene set enrichment analysis of the GSE12417-GPL96 dataset comparing GPR132 HIGH (top 10% of GPR132 expression) and GPR132 LOW (top 10% of GPR132 expression). I HL60 and MV4-11 cells were treated with 8GL (30 μM) and/or H89 (1 μM) for 72 h, and CD11b expression was detected using flow cytometry. Data are shown as mean ± SEM (n = 3). One-way ANOVA with Tukey’s multiple comparison tests was performed. **P < 0.01, ***P < 0.001. J Representative NBT reduction results of AML cell lines treated with or without 30 μM 8GL and/or 1 μM H89 for 72 h. The scale bar represents 10 μm.