Fig 1.
The plasmid map of pcDNA3.1.
Table 1.
The sequence of primers.
Fig 2.
Establishment and validation of DC-STAMP overexpression and knockdown THP-1 cell models.
(A) GSEA revealed significant enrichment of the mTORC1 pathway in DC-STAMP-high AML samples. (NES = 2.591, FDR q < 0.05, adjusted p = 0.008). (B) qRT-PCR validation of relative DC-STAMP mRNA expression levels in knockdown and overexpression THP-1 cells, normalized to the control group. (C) Western blot analysis of DC-STAMP protein expression in KD and OE groups (β-actin as loading control). Right panel: Quantitative densitometry of protein bands. (D) Flow cytometry was used to assess apoptosis in control and DC-STAMP-overexpressing THP-1 cells after 48 hours of treatment with different concentrations of vincristine. 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).
Fig 3.
The aberrant expression of DC-STAMP regulates apoptosis in AML cells.
(A) Cell viability of THP-1 cells in each group after 0h/24h/48h/72h of DC-STAMP knockdown or overexpression, measured using the CCK-8 assay. Data are normalized to the NC group (error bars represent mean ± SD). (B) Apoptosis levels assessed by Annexin V-FITC/PI double staining and flow cytometry after after 0h, 24h, 48h,72h of DC-STAMP knockdown or overexpression. Bar graph shows the total apoptosis rate (early + late apoptosis) in each group. 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).
Fig 4.
DC-STAMP exerts an upstream regulatory function on the mTOR signaling pathway.
(A) Immunofluorescence analysis of PI3K/AKT/mTOR pathway activation in THP-1 cells. Subcellular localization of phosphorylated AKT (p-AKT, Ser473, green) and phosphorylated mTOR (p-mTOR, Ser2448, red), with nuclear counterstaining by DAPI (blue). Scale bar: 20 μm. The lower panel shows quantitative analysis of fluorescence intensity (n = 3; error bars represent mean ± SD). (B) Expression levels of pro-apoptotic genes (Bax and Caspase-3), the anti-apoptotic gene Bcl-2, and pan-apoptotic/necroptotic/pyroptotic markers (RIPK3 and Caspase-1) were normalized to GAPDH (n = 3; error bars represent mean ± SD). (C) Western blot analysis of PI3K/AKT/mTOR signaling and apoptosis-associated proteins. Protein 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 served as the internal control. The right panel shows densitometric quantification of protein band intensity. 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).
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).
Fig 6.
High DC-STAMP expression in AML suppresses PANoptosis by upregulating the PI3K/AKT/mTOR signaling pathway, thereby accelerating leukemia progression.