Cxcl8 il 8

The CXCL1 antibody (9µg/mL; cat. no. MAB275; R&D SYSTEMS, Minneapolis, MN, USA), IL-8/CXCL8 (0.4µg/mL; cat. no. MAB208; R&D SYSTEMS, Minneapolis, MN, USA), and IL-18 (1.2µg/mL; cat. no. AF2548; R&D SYSTEMS, Minneapolis, MN, USA) were used for the neutralization assay. The normal mouse IgG1 antibody (1 µg/mL; cat. no. sc-3877; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) was used as the control antibody. The experiment was conducted in the same manner as the cell culture environment, and the subsequent experiments or results were performed/obtained after 24–48 h.

Antibodies against RanBP1 (cat. no. 8780; Cell Signaling Technology, Inc., Danvers, MA, USA), CD44 (cat. no. 3570; Cell Signaling Technology, Inc., Danvers, MA, USA), Sox2 (cat. no. 3579; Cell Signaling Technology, Inc., Danvers, MA, USA), Oct-4 (cat. no. 2750; Cell Signaling Technology, Inc., Danvers, MA, USA), Nanog (cat. no. 4893; Cell Signaling Technology, Inc., Danvers, MA, USA), β-actin (cat. no. sc-47778; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), ZEB1 (cat. no. sc-25388; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), SLUG (cat. no. sc-166476; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), SNAIL (cat. no. sc-10432; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), Twist (cat. no. sc-15393; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), ALDH1A1 (cat. no. ab6192; Abcam, Cambridge, UK), ALDH1A3 (cat. no. ab129815; Abcam, Cambridge, UK), E-Cadherin (cat. no. ab15148; Abcam, Cambridge, UK), CD133 (cat. no. ab19898; Abcam, Cambridge, UK), N-Cadherin (cat. no. 610920; BD Transduction, San Diego, CA, USA), Vimentin (cat. no. MA5-14564; Invitrogen, Waltham, MA, USA), CXCL1 (cat. no. PA5-115328; Invitrogen, Waltham, MA, USA), IL-8/CXCL8 (cat. no. MAB208; R&D SYSTEMS, Minneapolis, MN, USA), and IL-18 (cat. no. AF2548; R&D SYSTEMS, Minneapolis, MN, USA) were used for Western blot analysis and Immunocytochemistry assays.

ESCC cells were resuspended in RPMI-1640 without FBS and seeded at a density of 1.0 × 10⁶ cells/3 mL for TE-9 and TE-10 cells, and 1.7 × 10⁶ cells/3 mL for TE-11 cells in a 6-cm dish. The cells were cultured for 24 h and the culture supernatants were harvested as described in the cytokine array section. The Quantikine^® ELISA Human MMP9 (#DMP900, R&D Systems) or IL-8/CXCL8 (#D8000C, R&D Systems) immunoassay was used to measure the harvested culture supernatants. The culture supernatants of ESCC cells after STAT3 knockdown or treatment with recombinant human proteins were also processed as described above and used for MMP9 ELISA. The optical densities were measured using the Microplate Reader Infinite^® 200 PRO (Tecan, Männedorf, Switzerland).