Dcpib

HeLa or 3T3 fibroblasts (ATCC, Manassas, VA, USA) were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) either from Lonza BioWhittaker (Walkersville, MD, USA) or from Corning (Corning, NY, USA) supplemented with 10% fetal bovine serum (FBS, Fisher Scientific, Waltham, MA, USA) and antibiotics. To prepare hypoosmotic media, DMEM was diluted with water, or in some cases, with 10% FBS. Hyperosmotic media were prepared by adding sucrose to DMEM.
Osmolarity was not measured directly but estimated based on the manufacturer’s data for DMEM (340 mosm/kg for either source) and assuming ideal osmotic behavior. When 10% FBS, rather than water, was used to prepare dilutions, the numbers for osmolarity were adjusted accordingly.
The following reagents were used: gramicidin, fluorescein sodium, Hoechst 33258, DCPIB, and DiOC₁₈(3) (3,3′-dioctadecyloxacarbocyanine perchlorate; DiO), all from Sigma-Aldrich (St. Louis, MO, USA); torin 1 and rapamycin from ApexBio (Houston, TX, USA); 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS), amiloride, and cytochalasin D from Cayman Chemical (Ann Arbor, MI, USA); Acid Blue 9 (TCI America, Portland, OR, USA); NucView 488 (Biotium, Fremont, CA, USA); dextran Alexa 488 (Invitrogen, Eugene, OR, USA); tetramethylrhodamine ethyl ester (TMRE; Biotium, Hayward, CA, USA), and calcein AM ester (AnaSpec, Fremont, CA, USA).

They are STZ, angiotensin II, angiotensin-(1–7), losartan, PD123,319, hydroxocobalamin, L-NNA, 1400 W, apocynin, tiron, PEG-catalase, CDCF-DA, and DCPIB (Sigma, St. Louis, Mo., USA); DX600 (Anaspec Inc., Fremont, CA, USA); A779 (Bachem California Inc., Torrance, CA, USA); L-NPA (Tocris, Avonmouth, UK); DHE (Invitrogen, Carlsbad, CA, USA); ketamine (União Química, Jabaquara, SP, Brazil); xylazine (Calier Laboratory, Jubatuba, MG, Brazil); rabbit polyclonal anti-ACE2 antibody (Abcam, Cambridge, MA, USA); rabbit anti-angiotensin-(1–7) antibody (Phoenix Pharmaceuticals Inc., Burlingame, CA, USA); rabbit polyclonal anti-angiotensin-(1–7)-Mas receptor antibody (Alomone Labs, Jerusalem, Israel); biotinylated universal secondary antibody, avidin-biotin peroxidase complex (Vectastain Elite ABC kit, Universal, Vector Laboratories Inc. U.S. Headquarters, Burlingame, CA, USA).

DCPIB, NAC, 5-bromo-2-deoxyuridine (BrdU), MTT, 3-MA, antimouse and anti-rabbit secondary antibodies were purchased from Sigma-Aldrich (St. Louis, USA); LC3B, Beclin-1, GAPDH and LAMP2 primary antibodies were purchased from Genetex (Abcam, England); Antibodies against Beclin1 was obtained from Sigma-Aldrich (St. Louis, USA). Dihydroethidium (DHE) was from Molecular Probes (Eugene, OR, USA). LC3II primary antibody and Z-DEVD-FMK were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Terminal Deoxynucleotidyltransferase- mediated dUTP Nick End Labeling (TUNEL) was from Roche Applied Science (SandhoferStrasse, Mannheim, Deutschland).