Lamin b1

Proteins were extracted from cell lysis with the Radio Immunoprecipitation Assay (RIPA) buffer (Thermo Scientific, Carlsbad, California, USA) containing a protease inhibitor cocktail (Selleck, Huston, Texas, USA). BCA kit (Thermo Scientific, Carlsbad, California, USA) was used to quantify proteins and equal amounts was resolved by sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gel and transferred to the PVDF membrane (Millipore, Billerica, MA). The membranes were blocked for 2h with 5% skim milk at room temperature and incubated with primary antibodies at 4°C overnight with gentle shake. The PVDF membrane was then incubated with appropriate secondary antibody for 1 h at room temperature. The bands in the membranes were then detected using the enhanced chemo luminescence substrate kit (Thermo Scientific, Carlsbad, California, USA) and scanned with Image J software. The antibodies used in our study including MMP9, CCND1, XIAP, TWIST1, IκBα, p-IκBα, p-IKKα/β, IKKα, IKKβ, N-Cadherin, E-Cadherin, Vimentin, Lamin B1 and GAPDH are purchased from Cell Signaling (CST, Danvers, MA, USA).
Subcellular fractions were prepared from ESCC cells with a Minute™ Cytoplasmic & Nuclear Extraction Kits (Invent biotech, Eden Prairie, USA) according to manufacturer’s instructions.

TauCl as a crystalline sodium salt (MW 181.57) was prepared as described previously [12 (link)]. Primary antibody for protein modified with 4-hydroxynonenal (4-HNE) was purchased from Japan Institute for the Control of Aging (JaICA), Nikken SEIL Co., Ltd. (Shizuoka, Japan). Primary antibodies for cyclooxygenase-2 (COX-2), Signal transducer and activator of transcription (STAT3), phospho-STAT3^Y705, cyclin D1, α-tubulin, Kelch-like ECH-associated protein 1 (Keap1) and lamin B1 were provided by Cell Signaling Technology (Danvers, MA, USA), and those for Nrf2 and heme oxygenase-1 (HO-1) were supplied from Abcam (Cambridge, MA, USA) and Enzo Life Sciences (Farmingdale, NY, USA), respectively. Nuclear factor-κB (NFκB) p65 and phospho-NFκB p65 were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies for caspase-3 and cleaved caspase-3 were purchased from Cell Signaling Technology (Danvers, MA, USA).

Cell lysates were prepared using RIPA buffer (9806, Cell Signaling) supplemented with protease and phosphatase inhibitors (11836153001 and 4906845001; Roche) according to manufacturer's instructions. Lysates were mixed with 4× Loading Buffer (928‐40004, Li‐Cor) and denatured at 95°C for 5 min prior to electrophoresis. SDS‐PAGE was performed with the NuPAGE® electrophoresis system (Thermo Fischer) including 4%–12% Bis‐Tris gels (Thermo Fischer, # NP0336BOX) and MES SDS Running Buffer (NP0002; Thermo Fisher) according to the manufacturer's instructions. Western blotting onto nitrocellulose membranes (10600002; GE Healthcare) was performed inside an XCell II™ Blot Module (Thermo Fischer) for 1 h at 30 V constant (25 mM Tris‐BASE; 192 mM Glycine, 20% (v/v) Methanol transfer buffer). Membranes were washed with TBS (15 mM Tris–HCl pH 7.6; 136 mM NaCl) and blocked with TBS Blocking Buffer (927‐50000, Li‐Cor). Primary antibodies were incubated at 4°C overnight according to manufacturer's instructions. The following antibodies were used: Phospho‐Histone H2A.X (Ser139) (2577, Cell Signaling), Cyclin D1 (2978, Cell Signaling), Lamin B1 (13435, Cell Signaling), p16^Ink4a (80772, Cell Signaling), p21^Cip1 (2948, Cell Signaling), GAPDH (2118, Cell Signaling), FLAG (740001, Thermo Fischer), pMLC (3675, Cell Signaling), and alpha‐SMA (ab5694, Abcam).

Obacunone, hydrogen peroxide (H₂O₂), neomycin and puromycin were purchased from Sigma-Aldrich (St Louis, Mo). Antibodies for HO1 (#70081), NQO1 (#3187), Nrf2 (#12721), Keap1 (#8047), Tubulin (#2125) and Lamin B1 (#13435) were provided by Cell Signaling Tech (Shanghai, China). All cell culture reagents were from Gibco Co. (Suzhou, China).

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).