Lamin a

Cells were lysed in SDS lysis buffer (100 mM NaCl, 500 mM Tris, pH 8.0, 10% SDS). For detection of NF-κB p100/52, p65 and c-Rel, a nuclear extraction kit (Thermo Scientific, MA, USA) was used to separate nuclear and cytoplasmic fractions. Cell extracts were subjected to 8–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antibodies for Western blotting include: Galectin-3 (cat. 125402) (Biolegend, San Diego, USA), Galectin-1 (cat. Ab25138) and TSG101 (cat. ab30871; detects murine and human proteins) (Abcam, Cambridge, MA, USA), CD63, Erk1/2, NF-κB p65 (sc-8008 Figure 4G) (Santa Cruz Biotechnology, USA), phospho-Erk1/2, NF-κB p65 (cat. 8242) and calreticulin (cat. 2891S; detects murine and human proteins) (Cell Signaling Technology, USA), NF-κB p100/52 (cat. 06–413) (Millipore, USA). Gapdh (Chemicon International, USA or Millipore MAB374), α-tubulin (Oncogene Science, Cambridge, USA), lamin A, Histone H3 (Cell Signaling Technology, USA) or β-actin (cat. GTX109639, GeneTex) were used as a loading control.

ALO and recombinant human HMGB1 were purchased from Sigma Chemical Co. (St. Louis, MO), ERK signal pathway inhibitor, U1026 was purchased from Abcam (Cambridge, MA), specific antibody against HMGB1 (Product No. 6893S), RAGE (Product No. 6996S), TLR2 (Product No. 2229S), LaminA (Product No.4777 ) were purchased from Cell Signaling Technology (Danvers, MA), specific antibody against ERK1/2 (Product No. Ab54230), phosphorylated-ERK1/2 (Product No. Ab207470), PARP (Product No. Ab32138), cleaved-PARP (ab32561), BCL-2 (Product No. Ab32124), Caspase-3 (Product No. Ab184787), Cleaved Caspase-3 (Product No. Ab2302), TLR4 (Product No. ab13867) and β-actin (Product No. Ab8226) were purchased from Abcam (Cambridge, MA). HMGB1 ELISA kit (Product No. 326056538) was purchased from Shino-test Corporation (Japan). Annexin V/PI apoptotic kit (KGAV116) was purchased from KeyGen Biotech Co., Ltd. (Nanjing, China). BCA protein assay kit (Product No. P 0010) and CCK8 kit (Product No. C0037) were purchased from Beyotime Institute of Biotechnology (Haimen, China).

Polydatin was purchased from J&H Chemo Co., Hangzhou, China. RPMI-1640 medium, phosphate-buffered saline (PBS), fetal bovine serum (FBS), and trypsin-EDTA were purchased from Welgene Inc., Korea. Antibodies against NLRP3 (# 13158), phospho-p65 (# 3031), p65 (# 8242), Lamin A (# 4777), α-tubulin (# 2125), COX-2 (# 4842), caspase-1 (# 3866), SOD1 (# 4266), GPx (# 3206), HO-1 (# 43966), and β-actin (# 3700) were purchased from Cell Signaling Technology (Danvers, MA, USA). The ROS inhibitors N-acetyl-l-cysteine (NAC) and H₂DCFDA were purchased from Sigma Chemical Co., St. Louis, MO, USA.

Protein samples were separated by gradient electrophoresis on 8–15% SDS/PAGE gels. The following antibodies were used to detect the proteins of interest: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), LAMIN A, β-TUBULIN, SIRT3, Cytochrome c, Bax, Bcl-2, and Cytochrome c oxidase (COX) IV from Cell Signaling Technology (Danvers, MA, USA) and FoxO3a, SOD2, CAT from Abcam (Cambridge, UK). Western blot analysis was performed as described previously20 (link) using the protocols provided by the primary antibody suppliers.

HCECs were seeded in 60 mm dishes (6 × 10⁵ cells/mL) and co-treated with PCE (1, 10, 100 μg/mL) for 1 or 24 h. The cells were washed with phosphate-buffered saline (PBS) and lysed with RIPA lysis buffer (Invitrogen, Carlsbad, CA, USA). Cell lysate (30 mg) was separated by 8, 10, and 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to Polyvinylidene fluoride (PVDF) or nitrocellulose membranes. The membranes were blocked in skim milk dissolved in Tris-buffered saline with 0.1% Tween 20 (TBST) buffer for 1 h. Membranes were incubated with the following diluted (1:1000) primary antibodies: Cyclooxygenase-2 (COX-2), p65, phopho-p65, Poly (ADP-ribose) polymerase (PARP), Caspase-3, Bcl-2, Bcl-2-associated X protein (BAX), HO-1, Superoxide dismutase-1 (SOD-1), glutathione peroxidase (GPx) β-actin, and Lamin A (Cell Signaling, Danvers, MA, USA) in Tris-HCl-based buffer containing 0.2% Tween 20 (TBS-T; pH 7.5). Band intensities were measured using ImageJ software (NIH, Bethesda, MD, USA).

Pyrotinib (SHR1258) and dalpiciclib (SHR6390) were kindly provided by Hengrui Medicine Co., Ltd. Tamoxifen (HY-13757A) and trastuzumab were purchased from MCE company. Compounds were dissolved in dimethylsulfoxide (DMSO) at a concentration of 10 mM and stored at –20°C for further use. Trastuzumab were dissolved and used according to the manufacturer’s instructions. The following antibodies were purchased from Cell Signaling Technology (Beverly, MA): ER (#13258), p-HER2 (Tyr 1221/1222, #2243), HER2 (#4290), p-Akt (Ser473, #4060), AKT (#4685), p-mTOR (Ser2448, #5536), mTOR (#2983), pRb (Ser 780, #8180), Rb (#9309), CDK4 (#12790), CDK6 (#13331), Ubi (#3936), Lamin A (#4777), HSP90 (#4877), and GAPDH (#5174). The pCDK4 (Thr172, abs139836) antibody was purchased from Absin Technologies (Shanghai, China).