Hpa049259

The gastric cancer tissue samples (n=55) were formalin-fixed and paraffin-embedded. These tissue samples included 55 GC tissues and 29 matched tumor adjacent tissues. Immunohistochemical staining was then performed as previously described (22 (link),25 (link)). The sections were incubated with a rabbit monoclonal antibody (HPA049259, anti-ZNF382 antibody, 1:50 dilution; Sigma-Aldrich, St. Louis, MO, USA) at 4°C overnight. The following day, samples were rinsed with phosphate-buffered saline (PBS), incubated with rabbit secondary antibody (SP-9001, 1:100 dilution; ZSGB-BIO, Beijing, China) at 37°C for 30 min, stained with diaminobenzidine for 33 sec, and counterstained with hematoxylin for 5 sec to visualize the nuclei. Each section was assessed and scored by two independent pathologists who were blinded to the origin of all tissues. The widely accepted German semi-quantitative scoring criteria was used for scoring, and the staining index was determined by multiplying the score for staining intensity with the score for staining extent, as previously described (26 (link)). Clinical data included age, sex, histological type, differentiation grade and TNM stage. Clinical follow-up data for 55 patients were censored for the analysis.

The MKN45 and SGC7901 cells were cultured in 24-well plates with glass coverslips in the wells and transiently transfected with pcDNA3.1-Flag-ZNF382. Forty-eight hours later, the cells were fixed in 4% paraformaldehyde for 30 min, then permeabilized in 0.1% Triton X-100 for 5 min, followed by blocking with 1% bovine serum albumin (BSA) in PBS for 1 h. The cells were then incubated with primary antibodies against ZNF382 (HPA049259, 1:200 dilution; Sigma-Aldrich) and E-cadherin (sc-8426, 1:200 dilution) or Vimentin (sc-6260, 1:200 dilution) (both from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at 4°C overnight, then incubated with Alexa Fluor^® 594-conjugated (#A-11032, 1:200 dilution; Invitrogen; Thermo Fisher Scientific, Inc.) or FITC-conjugated (#111-585-003, 1:200 dilution; Jackson ImmunoResearch, West Grove, PA, USA) secondary antibody against rabbit or mouse IgG. 4′,6-Diamidino-2-phenylindole (DAPI) was used for nuclei counterstaining. Images were captured using a confocal laser scanning microscope (×200 and ×400 magnification).