Fig. 2
DPEP1 overexpression in colon cancer cells decreases NK cell cytotoxicity, whereas its knockdown enhances NK cell cytotoxicity. (A) Lysates of 13 colon cancer cell lines were subjected to SDS–PAGE followed by western blotting using an anti-DPEP1 antibody. (B) Cell proliferation assay. DPEP1-negative HCT116 and SW480 cells were transfected with DPEP1 or vector control, and cell growth was measured using the WST-1 assay. (C, D) DPEP1 knockdown enhances NK cytotoxicity. KM12C (C) or SW620 (D) cells were transfected with DPEP1 siRNA. Half of the cells were used to confirm knockdown by western blotting, and the remaining cells were stained with Calcein-AM, co-cultured with NK92 cells (E: T = 2:1), and assayed for Calcein release. (E, F) DPEP1 overexpression suppresses NK cytotoxicity. SW480 (E) or HCT116 (F) cells were transfected with DPEP1 plasmid. Western blotting confirmed DPEP1 expression. Calcein-AM–labeled cells were co-cultured with NK92 cells (E: T = 2:1), and Calcein release was measured. (G) Attenuation of NK cell cytotoxicity by NKp30 knockdown. NK92 cells were transduced with shRNA targeting NKp30 (shNKp30) or control shRNA (shCtrl), and knockdown was confirmed by western blotting. Calcein-AM–labeled colon cancer cells were incubated with NK92/shNKp30 or NK92/shCtrl cells (E: T = 2:1), and Calcein release was measured. 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; NS, not significant).
