P c fos

Erlotinib-resistant HCC827/ER cells were established as previously described [34 (link)] and cultured in a RPMI-1640 medium (Hyclone, Beijing, China, SH30809.01) supplemented with 10% FBS (R&S, Australia, 009106), penicillin and streptomycin (Gibco, New York, NY, USA, 15140122), and 5 μM Erlotinib (Selleck, Shanghai, China, s1023) at 37 °C and 5% CO₂. The parental HCC827 cell line was purchased from ATCC. The following antibodies were used: p-AKT^Ser473 (Cell Signaling Technology, Danvers, MA, USA, 4060), AKT (Abcam, Shanghai, China, ab79360), E-cadherin (Proteintech, Wuhan, China, 20874-1-AP), N-cadherin (ZEN Bio, Chengdu, China, 382812), vimentin (ZEN Bio, Chengdu, China, R22775), snail (Cell Signaling Technology, 3879), laminA/C (Santa Cruz, Starr County, TX, USA, sc-376248), laminB1 (Cell Signaling Technology, 12586), ZEB1 (Cell Signaling Technology, 3396), p-FGFR (Abcam, Shanghai, China, ab192589), p-ERK1/2 (Cell Signaling Technology, 4370), ERK1/2 (Proteintech, Wuhan, China, 67170-1-Ig), p-c-fos (Santa Cruz, TX, USA, sc-81485), c-fos (ZEN Bio, Chengdu, China, 340249), p-EGFR^Y845 (Cell Signaling Technology, 6963), and GAPDH (SAB, MD, USA, 48142).

The HDPCs (2 × 10⁵) were treated under different conditions for different time points. The EMPs (1 µM) were also added to the HDPCs as an extra control group. Then the cells were harvested and lysed in 200 µL lysis buffer (Beyotime, Shanghai, China) and centrifuged (12 000 rpm for 10 min, at 4°C). The protein concentration was evaluated with a BCA kit (Solarbio, Beijing, China). The protein was mixed with 5× loading buffer and heated at 95°C for 5 min, separated by precast PAGE gel (Beyotime, Shanghai, China) and transferred to polyvinylidene fluoride membranes. After blocked at room temperature for 15 min, then incubated with the primary antibody (DSPP, DMP-1, Smad2/3, c jun, p-c jun, c fos, p-c fos, c jun B) (Santa Cruz, California, USA), [p-ERK1/2, ERK1/2, alkaline phosphatase (ALP), p-Smad2/3, GAPDH] (Beyotime, Shanghai, China). Then, the membranes were incubated with the goat anti-rabbit IgG-HRP secondary antibody or goat anti-mouse IgG-HRP secondary antibody for 1 h, respectively. Then, an ultrasensitive chemiluminescence kit (Beyotime, Shanghai, China) was used to detect the protein bands. A chemiluminescence imager (Bio-Rad, California, USA) was used to scan the protein bands. The Image Lab software was used to analyze the relative protein expression levels.

3-[4,5-Dimethyl-2-yl]-2,5-diphenyl tetrazolium bromide (MTT), zerumbone (ZER) (purity ≥ 98%), 2′,7′-dichlorofluorescin-diacetate (DCFH₂-DA), LY294002 (PI₃K/AKT inhibitor), and N-acetylcysteine (NAC) were obtained from Sigma-Aldrich (St. Louis, MO). SB203580 (p38 MAPK inhibitor), PD98059 (ERK inhibitor), GF109203X (PKC inhibitor), and SP600125 (JNK inhibitor) were purchased from Calbiochem (La Jolla, CA). Compound C (AMPK inhibitor) and CKII (casein kinase II inhibitor) were obtained from Merck & Co., Inc. (Darmstadt, Germany). The following antibodies were purchased from their respective companies: p-c-Fos, p-c-Jun, Nrf2, Keap-1, β-actin [Santa Cruz Biotechnology Inc. (Heidelberg, Germany)]; histone [Cell Signaling Technology (Beverly, MA, USA)]; anti-HO-1 and anti-γ-GCLC [Gene Tex Inc. (San Antonio, TX, USA)]; and MMP-1, collagen type III [Abcam Inc. (Cambridge, MA, USA)]. All other chemicals, tissue culture, and common laboratory supplies were either purchased from GIBCO BRL (Grand Island, NY, USA) or Merck & Co., Inc. (Darmstadt, Germany) or Sigma-Aldrich (St. Louis, MO).