Journal: American Journal of Translational Research

Article Title: Protective effects of resveratrol on random-pattern skin flap survival: an experimental study

doi:

Figure Lengend Snippet: Resveratrol upregulated the level of autophagy in ischemic area of the flaps. On day 7, the level of autophagy in area II of flap was assessed by immunofluorescence for LC3II, IHC for CTSD, Western blotting for Beclin1, LC3II/I, CTSD, VPS34 and p62. A. Immunofluorescence for LC3II punctate dots in random skin flaps of the Control group and Rev group. Autophagosomes were labeled with LC3II (green), nuclei were labeled with DAPI (blue) (scale bar, 15 μm). B. LC3II-positive cell densities were quantified and analyzed in ischemic area in each group. C. IHC for CTSD expression in area II of flap in the Control group and Rev group (original magnification, × 200; scan bar, 50 μm). D. Optical density values of CTSD expression were quantified and analyzed in each group. E, F. Western blotting was performed for Beclin1, LC3II/I CTSD, VPS34 and p62 in the Area II tissue in the Control and Rev groups. The gels were run under the same experimental conditions, and cropped blots are used here. The Original images are available in Figure S1D. G. Optical density values of the expression of Beclin1, LC3II, CTSD, vps34 and p62 were quantified and analyzed in each group. Significance: *P < 0.05 and **P < 0.01 vs. the Control group. Data were expressed as means ± SEM, n = 6 per group.

Article Snippet: And other reagents and antibodies were from the following companies: rabbit monoclonal anti-GAPDH (AP0063; Biogot Technology, Shanghai, China). rabbit monoclonal anti-Matrix metallopeptidase9 (MMP9), anti-VEGF, anti-SOD1, anti-HO1, anti-caspase3 (CAPS3), anti-Phosphoinositide 3 kinase (VPS34) and anti-cathepsin D (CTSD) (19003-1, 10269-1, 12452-1, 10375-2, 10701-1, 213-27-1, and 19677-1; Proteintech Group, Chicago, IL, USA); rabbit monoclonal anti-cadherin5 (A0-2632-2; Boster Biological Technology, Wuhan, China). rabbit monoclonal anti-endothelial nitric oxide synthase (eNOS), anti-cytochrome c (CYC) and anti-Bax (11940S, 14796 and 32027; Cell Signaling Technology, Beverly, MA, USA); rabbit monoclonal anti-Microtubule-associated 1 protein light chain 3 (LC3) (L7543; Sigma-Aldrich Chemical Company, Milwaukee, WI, USA); mouse monoclonal anti-SQSTM1/p62 (ab56416; Abcam, Cambridge, UK); horseradish peroxidase (HRP)-conjugated IgG secondary antibody (Santa Cruz Biotechnology, Dallas, TX, USA); the BCA Kit (Thermo Fisher Scientific, Rockford, IL, USA); the Electrochemiluminescence (ECL) Plus Reagent Kit (PerkinElmer Life Sciences, Waltham, MA, USA); fluorescein isothiocyanate (FITC)-conjugated IgG secondary antibody (Boyun Biotechnology, Nanjing, China); 4’,6-Diamidino-2-phenylindole (DAPI) solution (Beyotime Biotechnology, Jiangsu, China).

Techniques: Immunofluorescence, Western Blot, Control, Labeling, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Puerarin alleviates apoptosis and inflammation in kidney stone cells via the PI3K / AKT pathway: Network pharmacology and experimental verification

doi: 10.1111/jcmm.70180

Figure Lengend Snippet: PUE attenuates COM crystal‐induced cell apoptosis. (A–F) Western blot and quantitative analysis of p‐PI3K, PI3K, p‐AKT (Ser 473), AKT, Cleaved‐caspase3, caspase3, Bax and Bcl2. (G, H) HK‐2 cells were stained with TUNEL for detection of apoptosis. The staining shows green fluorescence when the cells are in the process of dying. Data are shown as the mean ± S.E.M. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05.

Article Snippet: TNF‐α (26405‐1‐AP), IL‐1β (16765‐1‐AP), IL‐6 (21865‐1‐AP), AKT (10176‐2‐AP), p‐AKT (Ser 473) (28731‐1‐AP), PI3K (20584‐1‐AP), Bax (50599‐2‐Ig), Bcl‐2 (26593‐1‐AP) and Caspase3 (19677‐1‐AP) were obtained from Peprotech (Wuhan, China). p‐PI3K (#AF3242) was obtained from Affinity Biosciences(Jiangsu, China).

Techniques: Western Blot, Staining, TUNEL Assay, Fluorescence

Journal: Journal of Cellular and Molecular Medicine

Article Title: Puerarin alleviates apoptosis and inflammation in kidney stone cells via the PI3K / AKT pathway: Network pharmacology and experimental verification

doi: 10.1111/jcmm.70180

Figure Lengend Snippet: PUE protected against CaOx crystal‐induced renal apoptosis in mice. (A–F) Western blot and quantitative analysis of p‐PI3K, PI3K, p‐AKT (Ser 473), AKT, Cleaved‐caspase3, caspase3, Bax and Bcl2. (G) Representative immunohistochemistry images and quantitative analysis of p‐PI3K, PI3K, p‐AKT (Ser 473), AKT, Cleaved‐caspase3, caspase3, Bax and Bcl2. (H, I) Renal were stained with TUNEL for detection of apoptosis. Data are shown as the mean ± S.E.M. **** p < 0.0001, * p < 0.05.

Article Snippet: TNF‐α (26405‐1‐AP), IL‐1β (16765‐1‐AP), IL‐6 (21865‐1‐AP), AKT (10176‐2‐AP), p‐AKT (Ser 473) (28731‐1‐AP), PI3K (20584‐1‐AP), Bax (50599‐2‐Ig), Bcl‐2 (26593‐1‐AP) and Caspase3 (19677‐1‐AP) were obtained from Peprotech (Wuhan, China). p‐PI3K (#AF3242) was obtained from Affinity Biosciences(Jiangsu, China).

Techniques: Western Blot, Immunohistochemistry, Staining, TUNEL Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Puerarin alleviates apoptosis and inflammation in kidney stone cells via the PI3K / AKT pathway: Network pharmacology and experimental verification

doi: 10.1111/jcmm.70180

Figure Lengend Snippet: PUE exerted protective effects by PI3K/AKT pathway. (A–I) Western blot analysis of p‐PI3K, PI3K, p‐AKT (Ser 473), AKT, Cleaved‐caspase3, caspase3, Bax, Bcl2, IL‐1β, IL‐6 and TNF‐α in each group. (J, K) HK‐2 cells were stained with TUNEL for detection of apoptosis. (L‐O) The levels of superoxide anion and reactive oxygen species in HK‐2 cells were detected using DHE and DCF staining. Data are shown as the mean ± S.E.M. **** p < 0.0001, *** p < 0.001, ** p < 0.01.

Article Snippet: TNF‐α (26405‐1‐AP), IL‐1β (16765‐1‐AP), IL‐6 (21865‐1‐AP), AKT (10176‐2‐AP), p‐AKT (Ser 473) (28731‐1‐AP), PI3K (20584‐1‐AP), Bax (50599‐2‐Ig), Bcl‐2 (26593‐1‐AP) and Caspase3 (19677‐1‐AP) were obtained from Peprotech (Wuhan, China). p‐PI3K (#AF3242) was obtained from Affinity Biosciences(Jiangsu, China).

Techniques: Western Blot, Staining, TUNEL Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Puerarin alleviates apoptosis and inflammation in kidney stone cells via the PI3K / AKT pathway: Network pharmacology and experimental verification

doi: 10.1111/jcmm.70180

Figure Lengend Snippet: The influence of PI3K siRNA on the alleviation of COM‐induced apoptosis, inflammation and oxidative stress in HK2 cells by PUE. (A, B) Detection of transfection efficiency of Si‐PI3K by Western blot. (C–I) Western blot analysis of Cleaved‐caspase3, caspase3, Bax, Bcl2, IL‐1β, IL‐6 and TNF‐α in each group. (J–M) The levels of superoxide anion and reactive oxygen species in HK‐2 cells were detected by DHE and DCF. Data are shown as the mean ± S.E.M. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05.

Article Snippet: TNF‐α (26405‐1‐AP), IL‐1β (16765‐1‐AP), IL‐6 (21865‐1‐AP), AKT (10176‐2‐AP), p‐AKT (Ser 473) (28731‐1‐AP), PI3K (20584‐1‐AP), Bax (50599‐2‐Ig), Bcl‐2 (26593‐1‐AP) and Caspase3 (19677‐1‐AP) were obtained from Peprotech (Wuhan, China). p‐PI3K (#AF3242) was obtained from Affinity Biosciences(Jiangsu, China).

Techniques: Transfection, Western Blot