Human e cadherin

Hep-2 and Tu212 cells were inoculated into 6-cm dishes and exposed to different concentrations of pepsin in the culture medium. The cells were stimulated continuously for 2 h each day for 5 days. Total protein was extracted from the cells and the samples were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). The membranes were incubated overnight with rabbit monoclonal antibodies against human E-cadherin (1:1000; Cell Signaling Technology, USA), vimentin (1:100; Cell Signaling Technology), β-catenin (1:100; Cell Signaling Technology), snail (Cell Signaling Technology), and slug (Cell Signaling Technology) transcription factors and detected using chemiluminescence. An antibody specific for β-actin (1:10,000; Kangcheng Inc, Shanghai, China) was used as an internal control.

For the experiment involving the lymphocyte-mediated cytotoxicity to tumors, splenocytes were isolated from the humanized mice bearing PDAC cells. After lysing RBCs, the single-cell suspension was placed in the plate coated with human CD3 and CD28 antibodies. PDAC cells were prepared after 72 h of treatment with vehicle or siPD-L1@PLGA NPs (2 µg/mL). The activated splenocytes were co-cultured with siPD-L1@PLGA NP-treated PDAC cells at an E:T ratio of 20:1 and incubated at 37 °C for 24 h. The cells were collected and stained for human E-cadherin (Cell Signaling Technology, Danvers, MA, USA, 24E10), together with Annexin V (BD, cat no. 80-1729) and PI (ENZO, Farmingdale, NY, USA, cat no. 80-1731), followed by flow cytometry.