Anti jund

The esophageal squamous cell carcinoma (ESCC) cells KYSE140, KYSE520, and TE1 were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cells were subjected to mycoplasma examination and cytogenetically test and cultured with completed culture medium (basic medium supplemented with 10% fetal bovine serum) according to the standard protocols. The compound isoliquiritigenin was purchased from Selleck Chemicals (Houston, TX). Recombinant human EGF was product of R&D. The primary antibodies used in this study including anti-p-EGFR (Tyr1068) (#3777), anti-EGFR (#4267), anti-p-Akt (S473) (#4060), anti-Akt (#4691), anti-p-ERK1/2 (Thr202/Tyr204) (#4370), anti-ERK1/2(#4695), anti-c-Jun (#9165), anti-JunB (#3746), anti-JunD (#5000), anti-FosB(#4691), anti-c-Fos (#2251), anti-Fra1(#5281), anti-Cyclin D1(#55506), anti-β-actin (#3700), and anti-Histone H3 Ser10 (#53348) were products of Cell Signaling Technology (Danvers, MA). Lentivirus plasmids (pLKO.1-shEGFR) were purchased from Thermo Scientific (Huntsville, AL, USA).

MIA PaCa-2 and BxPC-3 cells were used to conducted to CHiP assays using the Chromatin Immunoprecipitation (ChIP) Assay Kit (Beyotime Biotechnology, Shanghai, China; P2078). A total of 10⁷ cells were cross-linked with 37% formaldehyde for 10 min at 37 °C and then neutralized using glycine. The cells were collected and treated with SDS lysis buffer containing 1 mM phenylmethylsulphonyl fluoride (PMSF) (Beyotime Biotechnology, Shanghai, China; ST506). Cell lysates were fragmented to 200–1000 bp by ultrasonication and mixed with ChIP dilution buffer. Lysates were incubated with anti-JunD (Cell Signaling Technology, Danvers, MA, USA; 5000; 1:200) or Rabbit IgG (Millipore, MA, USA; PP64B; 5 μg) overnight at 4 °C. The mixture was incubated with Protein A + G Agarose/Salmon Sperm DNA for 1 h at 4 °C, washed, and the immunoprecipitated complex was incubated with fresh elution buffer (1% SDS, 0.1 M NaHCO₃, and 5 M NaCl) at 65 °C for 4 h to reverse cross-linking. DNA purification was performed using Proteinase K and a PCR Clean-Up Kit (Beyotime Biotechnology, Shanghai, China; D0033). The extracted DNA samples were subjected to polymerase chain reaction (PCR) analysis. The ChIP primers are listed in Supplementary Table S1.