Fig. 3
Secreted DPEP1 attenuates NK cell cytotoxicity by binding to NKp30. (A) DPEP1 release via EVs. KM12C cells were cultured with or without the EV secretion inhibitor GW4869. Cell lysates, conditioned media, and EVs isolated from the media were analyzed by western blotting. CD9 and GAPDH were used as EV markers, and PARP1 as a control for cell lysates. (B) Interaction between secreted DPEP1 and NKp30. NK92 cells were treated with conditioned media from KM12C cells (± GW4869), and cell lysates were immunoprecipitated with anti-NKp30, followed by western blotting. (C) Flow cytometric analysis of NK92 cells treated with KM12C conditioned media and stained with anti-NKp30/APC and anti-DPEP1/FITC antibodies. (D) Confocal imaging showing colocalization of extracellular DPEP1 and NKp30 on NK92 cells treated with KM12C conditioned media (± GW4869). Cells were stained with anti-NKp30/FITC and anti-DPEP1/TRITC; DAPI was used for nuclear staining. Arrows indicate colocalization at the plasma membrane. (E) Increased NK cytotoxicity in response to DPEP1 knockdown. KM12C/shDPEP1 cells (± GW4869) were labeled with Calcein-AM, cocultured with NK92 cells, and Calcein release was measured. (F) Suppressed NK cytotoxicity by DPEP1 overexpression. HCT116 cells overexpressing DPEP1 (± GW4869) were labeled with Calcein-AM, cocultured with NK92 cells, and Calcein release was measured. (G) Conditioned media of KM12C cells (± GW4869, ±rDPEP1) were assessed as in (E) and (F). (H) Conditioned media from KM12C/shDPEP1 and shCtrl cells were immunoprecipitated with anti-DPEP1 or control IgG. The DPEP1-depleted media were then applied to NK92 cells, followed by NKp30 immunoprecipitation and western blotting. (I) DPEP1 depletion increases IFN-γ release from NK cells. NK92 cells were treated with DPEP1-suppressed or DPEP1-depleted conditioned media, and IFN-γ levels were quantified. Data represent mean ± SD from three independent experiments (n = 3), each performed in triplicate. Statistical significance was assessed using Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001; NS, not significant).
