Proliferating cell nuclear antigen pcna

Sodium butyrate (>99% purity) and cisplatin were obtained from Sigma-Aldrich (St. Louis, MO, United States). Sodium butyrate was dissolved in ultrapure water to prepare a 900 mM stock solution and cisplatin was dissolved in absolute dimethyl sulfoxide (DMSO) to prepare a 4 mg/ml (4 mg cisplatin +1 ml DMSO) stock solution, both of which were stored at −20°C.
Rabbit monoclonal antibodies against B-cell lymphoma 2 (BCL-2), BCL2 associated X (BAX), Cytochrome C (CytC), apoptotic protease activating factor-1 (Apaf-1), apoptosis inducing factor (AIF), proliferating cell nuclear antigen (PCNA), cleaved caspase-3, cleaved caspase-9, matrix metalloproteinase (MMP)-2, MMP-9, survivin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were obtained from Cell Signaling Technology (Danvers, MA, United States). The antibodies are diluted to a working concentration at a ratio of 1:1,000 and stored at 4°C. The secondary antibodies were used at a working concentration of 1:10,000 and were obtained from LI-COR (Lincoln, NE, United States).

Whole lysates were harvested using a Radioimmunoprecipitation Assay (RIPA) buffer containing protease inhibitor cocktail (Sigma-Aldrich), 1 mM phenylmethylsulfonyl fluoride, and phosphatase inhibitor cocktail set III (Calbiochem, CA, USA). Nuclear extracts were prepared using a nuclear extract kit (Active Motif, Carlsbad, CA, USA.). Equal amounts (20 μg) of protein were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Amersham Biosciences, Piscataway, NJ, USA), which were blocked with 5% skim milk in TBS/T (Tris buffered saline in 0.1% TWEEN® 20) buffer for 1 h. The membranes were then treated overnight with antibodies specific to iNOS, NF-κB p65, HO-1, NRF2, phospho-CREB, CREB, phospho-p38 (Thr180/Tyr182), p38, phospho-Erk (Thr202/Tyr204), Erk, phospho-JNK (Thr183/Tyr185), JNK, phospho-Akt (Ser473), and Akt (Cell Signaling Technology, Beverly, MA). Blots were incubated with horseradish peroxidase- (HRP-) conjugated secondary antibody for 1 h at room temperature. HRP was detected using a chemiluminescent detection reagent (Amersham Biosciences). β-actin (Sigma-Aldrich) and proliferating cell nuclear antigen (PCNA; Cell Signaling Technology) were used as loading controls. Chemiluminescence was visualized using an LAS-3000 LuminoImage analyzer (Fujifilm, Tokyo, Japan) [23 (link)].

Dexamethasone, nicotinamide, gentamicin, HEPES, NaCl, EDTA, glycerine, Triton-X, sodium fluoride, sodium orthovanadate, phenylmethanesulfonyl fluoride (PMSF), aprotinin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), acetaminophen (APAP), hydrogen peroxide (H₂O₂), carbon tetrachloride (CCl₄), and olive oil were obtained from Sigma Chemical (St. Louis, MO, USA). Dulbecco modified Eagle medium/Ham F12 (DMEM/F12), and Superscript III First-strand Synthesis System are products of Invitrogen (Carlsbad, CA, USA). Insulin, transferrin, and selenium (ITS) is from BD (Franklin Lakes, NJ, USA). Dimethylsulfoxide (DMSO) was obtained from Merck (Darmstadt, Germany). SYBR Green PCR master mix was obtained from Applied Biosystems (Warrington, UK). RNeasy mini kit is a product of Qiagen (Hilden, Germany). Phosphate-buffered saline (PBS) and all primers were synthesized by 1st BASE Oligos (Singapore). Primary antibodies were purchased from the following companies: phospho (Tyr705)-STAT 3, proliferating cell nuclear antigen (PCNA), NF-κB p65, cyclin D and cyclin E, Cell Signaling Technology (Danvers, MA, USA); hepatocyte growth factor (HGF), Abcam (Cambridge, UK). MSC-derived exosomes was prepared and purified as described previously [10 (link)].

Total proteins from Caco-2 cells transfected with miRNA mimics or control vector were extracted using RIPA lysis buffer containing PMSF and 1% protease inhibitor (Beyotime, China). The total protein concentrations were determined by BCA protein assay kit (Beyotime, China). Total protein samples (50 µg) were subjected to SDS/polyacrylamide gel electrophoresis and then transferred on to polyvinylidene fluoride membranes. The membranes were blocked with 5% non-fat milk in TBS + Tween 20 (TBST), and incubated at 4°C overnight with the following primary antibodies: c-Myc (Cell Signaling, U.S.A.), Proliferating cell nuclear antigen (PCNA) (Cell Signaling, U.S.A.), cyclin D1 (Santa Cruz, U.S.A.) and anti-β-catenin (Abcam, U.S.A.) antibody. β-actin (Abcam, U.S.A.) was used as the loading control. Horseradish peroxidase–conjugated secondary antibody were incubated for 1 h at room temperature and detected with an enhanced chemiluminscence kit (Beyotime, China). Finally, the expression levels of proteins were determined using Image-Pro Plus 6.0 software.

MET was purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China) and dissolved in phosphate-buffered saline (PBS) as a stock solution of 2 M. The NO prodrug JS-K was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) and dissolved in dimethyl sulfoxide (DMSO) as a stock solution of 5 mM. N-acetylcysteine (NAC) and glutathione disulfide (GSSG) were obtained from Beyotime Institute of Biotechnology (Shanghai, China) and dissolved in PBS to concentrations of 100 mM and 5 mM respectively. All stock solutions were stored at -20°C for further use. Antibodies against Bak, Bcl-2-associated X protein, B-cell lymphoma 2, caspase-3, caspase-9, cytochrome c (Cyto-C), Phosphorylated histone H2AX (γH2AX), DNA repair protein Rad51, and Proliferating cell nuclear antigen (PCNA) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA), and antibody against GAPDH was purchased from Abcam (Cambridge, UK). Horseradish peroxidase-conjugated IgG secondary antibodies were purchased from EarthOx Life Sciences (Millbrae, CA, USA).