Anti e cadherin e cad

RPMI-1640 and fetal bovine serum (FBS) were obtained from ThermoFisher Scientific (Cleveland, OH, USA). Anti-S100A7, anti-Stat3 and anti-phosphorylated Stat3 (anti-p-Stat3) antibodies were obtained from GeneTex (Irvine, TX, USA). The anti-Src antibody was acquired from Cell Signaling Technology (Danvers, MA, USA). Anti-p-Src (Y418), anti-Twist and anti-E-cadherin (E-cad) antibodies were obtained from Abcam (Cambridge, UK). Anti-glyceraldehyde3-phosphate dehydrogenase (GAPDH) and anti-β-actin antibodies were purchased from Santa Cruz (Capitola, CA, USA). The polymerase chain reaction (PCR) forward and reverse primers were purchased from Purigo Biotech (Taipei, Taiwan). Luteolin (Lu) was purchased from Toronto Research Chemicals (North York, ON, Canada). Quercetin (Qu) was purchased from Nacalai Tesque (Kyoto, Japan). Su6656, S3I-201, dimethyl sulfoxide (DMSO), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (Merck, Darmstadt, Germany).

Cells were cultured on sterilized glass slides in 24-well plates, treated with pre-cooled paraformaldehyde (4%) for 30 min and Triton X-100 (0.1%) at room temperature for 10 min, blocked with 1% BSA for 30 min, then incubated with primary antibody at 4°C overnight, anti-MLKL (1:100, Abcam), anti-E-cadherin (E-cad) (1:500, Abcam), anti-CD86 (1:100, Abcam) and anti-CD206 (1:100, Abcam). Finally, slides were incubated with fluorescence labeled secondary antibody (1:1,000) for 1 h at room temperature, then stained with DAPI for 5 min, and mounted with antifade mounting medium. Slides were observed with a fluorescence microscope.