Sirt3

HT (purity ≥98%) was obtained from Extrasynthese (Lyon, France). Dulbecco's modified Eagle's medium (DMEM) was from BiochromAG (Berlin, Germany). RNA was isolated using the RNeasyPlus Mini kit (Qiagen, Hilden, Germany) and cDNA was prepared with Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Fermentas Intl., Vilnius, Lithuania). Real-time PCR was performed using SYBR Fast Master Mix (2x) Universal (KAPABiosystems, Massachusetts, USA). Primers were synthesized by Biomedal S.L. (Sevilla, Spain). Nrf2 siRNA (sc-37049), scramble siRNA (scr siRNA) and the transfection reagent were from Santa Cruz Biotechnology (CA, USA). Opti-MEM reduced serum medium was bought from Thermo Fisher Scientific. α-Tubulin antibody, foetal bovine serum (FBS), sulforhodamine B (SRB), trichloroacetic acid (TCA), 2´,7´-dichlorofluorescin diacetate (DCFH-DA), glutathione reductase (GR), reduced glutathione, NADPH, cumene hydroperoxide, and other general reagents were from Sigma (St. Louis, MO, USA). Heme oxygenase-1 (HO-1) was measured with the enzyme linked immunosorbent assay (ELISA) kit CSB-E08266h from Cusabio (Barksdale, USA). Primary antibodies (PGC-1α, Nrf2, ERRα, SIRT3) were purchased from Santa Cruz Biotechnology, Inc., (CA, USA) except anti-α-tubulin (Sigma, St. Louis, Mo, USA).

RIPA lysis buffer (Beijing Dingguo Technology) containing phosphatase and protease inhibitors was used to extract total protein the differentiated cells after 8 days of treatment. The protein concentrations were quantified using a BCA kit (Beijing Dingguo Technology). Forty micrograms of the extracted proteins were subjected to SDS-PAGE (110 V, 2 h), followed by transferring (110 V, 90 min) onto polyvinylidene difluoride membranes (Millipore, USA). After blocking with 5% skimmed milk at RT for 1 h, the membranes were incubated with primary antibodies as follows: AMPK (rabbit polyclonal, 1:1000, #sc-61, Santa Cruz), P-AMPK (rabbit polyclonal, 1:1000, #sc-61, Santa Cruz), microtubule-associated protein 3 (LC3 rabbit polyclonal, 1:1000, #sc-61, Santa Cruz), P62 (rabbit polyclonal, 1:1000, #sc-61, Santa Cruz), Sirt1 (mouse monoclonal, 1:500, #sc-7273, Santa Cruz) and Sirt3 (rabbit polyclonal, 1:1000, #sc-61, Santa Cruz) at 4 °C overnight. After rinsing with TBST buffer, the membranes were incubated with the corresponding HRP-conjugated secondary antibodies (DAKO, Denmark) at RT for 45 min. After washing 3 times with TBST buffer, the protein blots were visualized with ECL kit (Beijing Dingguo Technology). Next, the membranes were detected using an enhanced chemiluminescence detection system (Pierce, Rockford, USA).

Renal cortex proteins were homogenized with a lysis buffer containing: 50 mM HEPES pH 7.4, 250 mM NaCl, 5 mM EDTA, 0.1% NP-40, and complete protease inhibitor (Roche, No. Cat. 11697498001). Protein concentration was evaluated by the Lowry Protein Assay (Bio-Rad, Cat No. 5000113 and 5000114, Hercules, CA, USA). Proteins obtained from each animal were electrophoresed using 20 µg of proteins on an 8.5% acrylamide denaturing gel containing SDS. The membranes were incubated overnight at 4 °C with the primary antibody Sirt7 (Santa Cruz, Cat. No. sc365344, 1:1000, Dallas, TX, USA), Sirt3 (Santa Cruz, Cat. No. sc-365175, 1:1000, Dallas, TX, USA), IL-6 (Santa Cruz, No. Cat. Sc57315, 1:1000, Dallas, TX, USA), NFκB p65 total (F-6, Santa Cruz, No. Cat. sc-8008, 1:1000, Dallas, TX, USA), p-NFκB p65 (27.Ser 536, Santa Cruz, No. Cat. sc-136548, 1:1000, Dallas, TX, USA), Pol II (F-12, Santa Cruz, No. Cat. sc-55492, 1:1000, Dallas, TX, USA), and HRP β-actin antibody [AC-15] (Abcam, Cat. No. ab49900, 1:1,000,000, Cambridge, UK). Three 10 min washes were performed with 1x TBS-Tween, and then incubated with the secondary antibody coupled to HRP, anti-rabbit, or anti-mouse IgG (Santa Cruz, Cat. No. sc-2031 or sc-2004, respectively 1:5000, Dallas, TX, USA). Tissue proteins evaluated by Western blot were normalized by detection of β-actin.

Extracted proteins were separated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane. The membrane was blocked with 5% nonfat milk and incubated with primary antibodies for β-actin (Proteintech, 81115-1-RR, 1:5000), ALDH1L2 (Proteintech, 21391-1-AP, 1:1000), acetylation (Cell Signaling Technology, 9441S, 1:1000), Flag (MBL, M185–3, 1:5000), SIRT3 (Santa cruz, sc-365175, 1:1000), HA (Proteintech, 51064-2-AP, 1:5000), His-Tag (Abmart, 2A8, 1:1000)