Ang 2

Differentiated podocytes were treated with 1 μM Ang II (Tocris Bioscience) for 24 h. To examine the beneficial effects of losartan on Ang II-induced podocyte injury, differentiated cells were incubated with culture media containing 1 μM losartan (DuPont 753, Merck Pharmaceuticals) for 30 min, followed by incubation with Ang II plus losartan for 24 h. To inhibit NHE1 and p38 MAPK activity, cariporide (10 μM, Santa Cruz Biotechnology, Dallas, TX, USA) and SB203580 (0.1 μM, Merck Millipore, Temecula, CA, USA) were added for 30 min, followed by incubation with Ang II plus specific inhibitors for 24 h.

Western blotting was performed as described [47 (link)] with rabbit polyclonal anti-ENO1, LDHA antibody (1:1,000; Proteintech, USA), anti-cyclin D1, p21, cyclin E1, C-Myc antibody (1:1,000; Epitomics, Burlingame, USA), anti-CDK4 antibody (1:400; Santa Cruz Biotechnology, Santa Cruz, USA), anti-pRb (Ser780), FAK, p-FAK (Tyr397), AKT, p-AKT (Ser473), PI3K, p-PI3K (Tyr458), snail, β-catenin, N-cadherin, vimentin, and E-cadherin antibody (1:1,000; Cell Signaling Technology, Danvers, USA). An HRP-conjugated anti-rabbit IgG antibody was used as the secondary antibody (Zhongshan, Beijing, China). Signals were detected using enhanced chemiluminescence reagents (Pierce, Rockford, IL). Ang II was purchased from the Santa Cruz Biotechnology (Santa Cruz, USA).

Polyclonal antibodies against p-Akt (Ser473), Akt, and p-GSK3β, and monoclonal antibodies to β-catenin and Vimentin, were purchased from Cell Signaling Technology (Danvers, MA, USA). Polyclonal antibodies to JNK, Slug, Sox2, p-Stat6, Stat6, Ang-2, and VEGF, and monoclonal antibodies to p-JNK, β-actin, and Oct4 were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Polyclonal antibody to Snail was purchased from Novus Biologicals (Littleton, CO, USA). The monoclonal antibody E-cadherin was obtained from BD Transduction Laboratories (San Jose, CA, USA). The polyclonal antibody Twist was purchased from Abcam Inc. (Cambridge, MA, USA). Monoclonal antibodies to IL-4 and IL-4Rα were obtained from R&D systems (Minneapolis, MN, USA). Anti-mouse and anti-rabbit Alexa Fluor 488 and 555-conjugated secondary antibodies were purchased from Thermo Fisher scientific (Waltham, MA, USA). Inhibitors of JAK, PI3K (LY294002), and JNK (SP600125) were purchased from Merck Millipore (Darmstadt, Germany). Recombinant IL-4 was obtained from R&D systems (Minneapolis, MN, USA).

Fetal bovine serum (FBS) and trypsin (0.25% w/v) were purchased from Hyclone (Thermo Fisher Scientific Inc. Rockford, IL). Topotecan (TOPO) was purchased from 21st Century Global E-Commerce Network (East Sussex, UK). Dimethyl sulfoxide (DMSO), sulforhodamine B (SRB), TRIS buffer, acetic acid, ECL western blotting substrate for chemiluminescence were obtained from Bio-Rad (Hercules CA, United States ). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and RNase A were purchased from Sigma-Aldrich Inc (St. Louis, MO). Mouse anti-human antibodies (MMP-9, MMP-1, Ang-2, VEGF, Maspin and c-Fos were purchased from Santa Cruz Biotechnology (uPA; H77A10, MMP-1; SB12e, Ang-2; F-1, VEGF; JH121, Maspin; E10 Santa Cruz, CA) and Cell Signaling Technology (PAI-1; D9C4, uPAR; D7X2N, MMP-9; D6O3H, c-Fos; 9F6, Danvers, MA). Goat anti-Rabbit IgG (H = L) Secondary antibody, HRP, was purchased from Cell Signaling Technology; 345,897 (Danvers, MA). β-actin was purchased from Sigma-Aldrich; A5316. All glass and plasticware were purchased from VWR (Radnor, PA).

Samples harvested from the wounds were fixed with 4% paraformaldehyde (PFA) and embedded in paraffin. Serial 5-μm sections were cut and stained with hematoxylin and eosin (HE) for observation of histological changes in the burn wounds.
For immunohistochemical staining, a panel of primary antibodies against rat Ang-1 (Santa Cruz Biotechnology, 1∶200), Ang-2 (Santa Cruz Biotechnology, 1∶150), Tie-2 (Santa Cruz Biotechnology, 1∶200), VEGF (Abcam, 1∶150), MMP-2 (Abcam, 1∶300), and MMP-9 (Abcam, 1∶200) were used. The activity of endogenous peroxidase was quenched by incubation with hydrogen peroxide (3%) for 5 min, followed by microware treatment at 500 W for 5 min in citrate buffer for antigen retrieval. Next, the sections were incubated with primary antibodies at 4°C overnight. After thorough washing, the sections were incubated in goat anti-rabbit antibody (Invitrogen, 1∶500) for 30 min, followed by incubation in avidin-biotin complex (Elite ABC kit; Vector Laboratories) for 30 min. Color was developed in 3′ 3-diaminobenzidine (Dako), and nuclei were stained with hematoxylin (Sigma). Negative control staining experiments were performed by omission of the primary antibody. Images were captured using a light microscope (Olympus BX51 WI).

The following inhibitors or reagents were used in this study: LY294002 (Enzo Life Sciences, USA), MK-2206 (Invitrogen, USA), C3 exoenzyme (Cytoskeleton, USA), and pertussis toxin (PTX; Invitrogen, USA). YAP and phospho-YAP (Ser127) were purchased from Cell Signaling (Cell Signaling technology, USA). CTGF, ANG-2, and GAPDH antibodies were from Santa Cruz Biotechnology (Santa Cruz, USA). Alexafluor secondary antibodies were from Life Technologies, USA.