Lamin b

In brief, 40 μg protein was loaded to SDS-PAGE, and then, the PVDF membrane was used to transfer the protein. Membranes were blocked in 5% skim milk for 1 h. Antibodies against p65 (1 : 1000, Cell Signaling Technology, Beverly, USA), lamin B (1 : 1000, Cell Signaling Technology, Beverly, USA), and GAPDH (1 : 1000, Beyotime Biotechnology, Shanghai, China) were used to incubate the membranes for a whole night. Next day, the membrane was incubated with a HRP-conjugated secondary antibody for 1 h. The blots were visualized using the chemiluminescent detection kit (Pierce, Thermo Scientific, Waltham, USA).

PDE3A (catalog number A302-740A, 1:1000 [Bethyl Laboratory]; catalog number Sc-293446, 1:20 [Santa Cruz Biotechnology]), Flag (catalog number F7425, 1:2000; Sigma), GAPDH (catalog number 60004-1-1g, 1:5000; ProteinTech), Anti-phospho-PKA substrate antibody (catalog number 9621S or 9624, 1:1000; Cell Signaling), SMAD4 (catalog number 46535, GTX01674; Cell Signaling) GATA4 (GATA4 binding protein 4; catalog number 36966, 1:1000; Cell Signaling), HDAC-1 (catalog number 34589, 1:1000; Cell Signaling), lamin B (catalog number 12586, 1:1000; Cell Signaling), lamin A/C (catalog number 4777S, 1:1000; Cell Signaling), actin-1 alpha (catalog number 17521-1-AP; Proteintech), RFP (catalog number ab62341, 1:2000; Abcam), GFP (catalog number ab6556, 1:1000; Abcam), horseradish peroxidase–conjugated goat anti-mouse (catalog number sc2005, 1:5000 [SCBT]; catalog number 705-035-151 [Jackson ImmunoResearch Labs]) or anti-rabbit secondary antibodies (catalog number sc2030, 1:5000 [Santa Cruz Biotechnology]; catalog number 705-035-152 [Jackson ImmunoResearch Labs]), antibodies conjugated with Alexa-Fluor 647 (catalog number A-21235, 1:250; Thermo Fisher Scientific), Alexa-Fluor 488 (catalog number A-11008, 1:250; Thermo Fisher Scientific), and wheat germ agglutinin conjugated with Alexa 488 (catalog number W11261; Invitrogen).

The LPS (Escherichia coli 0127:B8), dimethyl sulfoxide (DMSO), PMA (phorbol 12-myristate 13-acetate) were purchased from Sigma-Aldrich (St, Louis, MO, USA). MEM medium, RPMI 1640 medium, fetal bovine serum (FBS), penicillin/streptomycin and phosphate-buffered saline (PBS) were purchased from Gibco Life Technologies (Grand Island, NY, USA). The Griess Reagent System was obtained from promega (Promega, Madison, WI, USA). Enzyme-linked immunosorbent assay (ELISA) kits for TNF-α, IL-6, and PGE2 were purchased from R&D Systems (Minneapolis, MN, USA). IL-1β, TNF-α, IL-6 and GAPDH primers were purchased from Bioneer (Daejeon, Korea). Tin protoporphyrin IX (SnPP) was purchased from Porphyrin Products (Logan, UT, USA). Primary antibodies, including iNOS, COX-2, p65, Lamin B, phospho-p65, IκBα, phospho-ERK, ERK, phospho-p38, p38, phospho-JNK, JNK, HO-1 and Nrf2 were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Primary monoclonal anti-β-Actin antibody was purchased from Sigma-Aldrich (St, Louis, MO, USA). The secondary antibody was purchased from Jackson ImmunoResearch Laboratories, Inc.

Licochalcone A (Lico A), purity > 98%, was provided by the Chengdu Herbpurify, Co., Ltd. (Chengdu, China). 3-(4, 5-dimethylthiazol-2-y1)-2, 5-diphenyltetrazolium bromide (MTT), Acetaminophen, t-BHP, and DMSO were purchased from Sigma–Aldrich (St. Louis, MO, United States). Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), Penicillin and streptomycin were acquired from Invitrogen-Gibco (Grand Island, NY, United States). Antibodies against p-JNK, JNK, Keap1, Nrf2, HO-1, NQO1, GCLC, GCLM, cytochrome c, Bax, Bcl-2, caspase 3, Lamin B, β-actin were obtained from Cell Signaling (Boston, MA, United States) or Abcam (Cambridge, MA, United States). Additionally, ALT, AST, MDA, MPO, GSH, and SOD test kits were supplied by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The GSH and GSSG test kit was offered by Beyotime Biotechnology (Shanghai, China). All other chemicals were offered by Sigma–Aldrich (St. Louis, MO, United States), if not otherwise indicated.

MTT powder (1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan), RIPA buffer, TRIZOL reagent, CoCl₂, DCF-DA, and Protoporphyrin IX (SnPP) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). AnnexinV/PI apoptosis assay kit was obtained from BD Biosciences (San Diego, CA, USA). Antibodies against Caspase3, Caspase7, Caspase8, Caspase9, HIF1α, p53, VEGF, GLUT1, SOD, CAT, HO-1, and LaminB were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-Bcl-2, anti-β-actin, and anti-Nrf2 antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). The RT PreMix kit was purchased from Enzynomics (Daejeon, Korea). SYBR Premix Ex Taq was obtained from Takara (Shiga, Japan). Nuclear and Cytoplasmic Extraction Reagents Kit (NE-PER) and ECL Western blot detection reagents were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

Total cell lysates were prepared using cell lysis buffer containing protease inhibitor cocktail (Beyotime Biotechnology, Shanghai, China). Nuclear and cytoplasmic proteins were extracted using a commercial kit (Beyotime Biotechnology, Shanghai, China). Equal amounts of total protein were separated by SDS-PAGE, and electrically transferred to PVDF membranes (Millipore, Bedford, MA, USA). After blocking with 5% non-fat milk, the membranes were incubated with primary antibody for STAT3, phospho-STAT3^Tyr705, mTOR, phospho-mTOR^Ser2448, eIF4E, eIF4G, phospho-4EBP1^Thr70, Caspase3, PARP, γ-H2AX^Ser139, Lamin B, c-Myc, Survivin, Bcl-2 (Cell Signaling Technology, Danvers, MA, USA) and β-actin, 4EBP1 (Abcam, Cambridge, UK). Next, the membranes were washed with Tris-buffered saline with Tween 20 (TBST) buffer 3 times and incubated with horseradish peroxidase-conjugated second antibody. Immunoblotting signals were detected using an enhanced chemiluminescence method.