DC-STAMP activates the PI3K/AKT/mTOR signaling pathway to regulate PANoptosis in acute myeloid leukemia
Fig 5
Inhibition of the downstream PI3K rescues DC-STAMP activation-induced malignant proliferation in AML.
(A) Cell viability of THP-1 cells in each group and different time points, measured using the CCK-8 assay. Data are normalized to the negative control group (NC) (n = 3; error bars represent mean ± SD). (B) Immunofluorescence analysis of PI3K/AKT/mTOR pathway activation. Subcellular localization of phosphorylated AKT (p-AKT, Ser473, green) and phosphorylated mTOR (p-mTOR, Ser2448, red), with nuclei stained by DAPI (blue). Scale bar: 20 μm. The lower panel shows quantitative analysis of fluorescence intensity (n = 3; error bars represent mean ± SD). (C) Western blot analysis of PI3K/AKT/mTOR signaling and apoptosis-related proteins. Expression levels of PI3K, p-PI3K (Tyr458), mTOR, p-mTOR (Ser2448), AKT, p-AKT (Ser473), and apoptosis regulators Bcl-2, Bax, and Cleaved Caspase-3 were detected. β-actin was used as the loading control. The lower panel presents densitometric quantification of protein band intensity (n = 3; error bars represent mean ± SD). (D) Apoptosis levels assessed by Annexin V-FITC/PI double staining and flow cytometry in the Vehicle control, DC-STAMP overexpression (DC-STAMP-OE), DC-STAMP-OE + 10 μM LY294002 (DC-STAMP-OE + LY), and Empty control + 10 μM LY294002 (Empty+PI3K-LY) groups. The lower panel shows quantitative results of total apoptosis rate (early + late apoptosis). Each result represents the mean of three independent biological replicates. Error bars represent mean ± SD). *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed t-test).