Complete lysis m

All animal studies were performed under a protocol approved by the Institutional Animal Care and Use Committee at Wright State University. Six‐week‐old male db/db diabetic mice (C57BL/KsJ (BKS.Cg‐Dock7m +/+ Lepr^db/J) and their age‐matched nondiabetic lean control mice (db/m) were obtained from Jackson Laboratories (Bar Harbor, ME, USA). Mice were housed individually in cages with ad libitum access to food and water and maintained at room temperature (22°C) with 12:12 hr light‐dark cycles. For tissue acquisition for histology, mice were deeply anesthetized with sodium pentobarbital (60 mg/kg, administered intraperitoneally) and then perfused transcardially with cold PBS and 4% paraformaldehyde. Every effort was made to minimize suffering and pain. For tissue acquisition for western blot, ELISA and enzyme activity, mice were euthanized by decapitation and kidneys were dissected and stored at –80°C.
Kidney tissues from NEP gene knockout mice (NEP^−/−) and their littermate wild‐type (NEP^+/+) were obtained from Dr. Bao Lu (Harvard Medical School). The kidney tissues were homogenized on ice in phosphate‐buffered saline (PBS) containing protease inhibitor (Complete lysis M, Roche diagnostics). Homogenates were then centrifuged at 10,000g for 10 min at 4°C to remove cellular debris. The supernatants were collected, aliquoted, and stored at (−80°C).

Protein from cell lines was isolated with M-PER Mammalian Protein Extraction Reagent (78501, Thermo Scientific, MA, USA). Protein from prostate cancer tissue was extracted with Complete Lysis-M (13354520, Roche, Mannheim, Germany). Protein concentration was determined by the BCA method (23225, Beyotime, Shanghai, China). Proteins were separated using 6-12% SDS-PAGE and transferred onto nitrocellulose membrane (10401396, GE healthcare, OH, USA). The membranes were blocked by blocking buffer (927-40000, Odyssey, MA, USA) for 1 hour and then incubated in the primary antibodies at 4°C overnight. After washing with TBST, the membranes were incubated with secondary antibodies at room temperature for 1 hour. Protein bands were visualized by infrared imaging system (Odyssey) and quantified with Odyssey application software. Rabbit anti-LC3B, SQSTM1, ATG7, MTOR, p-P70S6k, and cleaved CASP3 antibodies were purchased from Cell Signaling Technology, MA, USA (2775, 5114, 8558, 2983, 9208, 9664). Anti-GAPDH antibody was purchased from Abcam, Cambridge, UK (9485).

Mouse monoclonal anti-β-actin, rabbit polyclonal anti-bcl-2, rabbit polyclonal anti-Puma, rabbit polyclonal anti-cleaved-Bid, rabbit polyclonal anti-Bak, rabbit polyclonal anti-cleaved PARP, rabbit polyclonal anti-p53 and rabbit polyclonal anti-P21 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase linked secondary sheep anti-mouse and rabbit anti-horse IgG were obtained from GE Healthcare (Chicago, IL, USA). Propidium iodide (PI) and Annexin V were purchased from BD Pharmingen (Franklin Lakes, NJ, USA). Complete lysis-M was purchased from Roche Applied Science (Indianapolis, IN, USA). AO/EB nuclear dyes were purchased from Beyotime (Shanghai, China). Curcumin, niacin, 3-[4–dimethylthiazol-2-yl]-2, 5-diphenyltetrazoliumbromide (MTT) and other chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA). Curcumin nicotinate (CN) was in-home synthesized. For experimental uses, CN was dissolved in DMSO at 10 mM as stock solution and then diluted to concentrations indicated.

After 35 min of stimulation THP-1 macrophages with LPS, the medium was aspirated, and the cells were washed with ice-cold PBS twice. Afterwards, the cells were lysed with cOmplete™ Lysis-M (Roche, Basel, Switzerland) containing Protease and Phosphatase inhibitor cocktail (Roche, Basel, Switzerland) and centrifuged at 14,000g for 15 min at 4 °C. For NFκB p65 nuclear translocation after LPS stimulation the cells were lysed using Mammalian Nuclear and Cytoplasmic Protein Extraction Kit (SERVA, Heidelberg, Germany). The total protein concentration of the cell and nuclear lysates was quantified with BCA protein assay kit (Bio-Rad, Hercules, CA, USA) using BSA as a standard. After equilibration of protein concentration between the samples, the lysates were boiled with 4xLaemmli Sample Buffer (Bio-Rad) and stored in − 20 °C.

Cells were incubated for 5 min in lysis buffer (Complete lysis M; Roche Diagnostics, Basel, Switzerland). The protein concentration in the lysate was measured using a BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA). Equal amounts of protein (20 μg) were dissolved in NuPAGE LDS sample buffer (Invitrogen) and a 10% sample reducing agent (Invitrogen). The lysates were boiled at 70 °C for 10 min and then loaded into and subjected to electrophoresis in NuPAGE 4–12% Bis-Tris gels (Invitrogen) at 200 V for 60 min. The proteins were then transferred onto polyvinylidene difluoride membranes (Invitrogen), and the membranes were blocked with WesternBreeze Blocker/Diluent (Invitrogen). The membranes were then probed with the anti-FLG antibody, anti-OVOL1 antibody, and a mouse monoclonal antibody against human β-actin (anti-β-actin) (Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C. Horseradish peroxidase-conjugated anti-mouse IgG antibodies (Cell Signaling Technology) served as a secondary antibody. The visualization of protein bands was accomplished with the SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) by ChemiDoc touch imaging system (Bio-Rad).

RNeasy mini kit was purchased from Qiagen (Valencia, CA, USA). qPCR reagents SuperScript^® VILO™ cDNA synthesis kit, TaqMan^® Fast Advanced Master Mix, AKR1C1 and AKR1C2 TaqMan^® Gene Expression Assay were obtained from Life Technologies (Carlsbad, CA, USA). Complete Lysis-M was obtained from Roche (Indianapolis, IN, USA). Pierce ECL Western Blotting Substrate and Restore Western Blot Stripping Buffer were purchased from Thermo Fisher Scientific (Rockford, IL, UA). NIC (nicotine) was obtained from Sigma Chemical Company (St. Louis, MO, USA).