Journal: Brazilian Journal of Medical and Biological Research

Article Title: Ginsenoside Rg1 protects human renal tubular epithelial cells from lipopolysaccharide-induced apoptosis and inflammation damage

doi: 10.1590/1414-431X20176611

Figure Lengend Snippet: A , Structural formula of ginsenoside Rg1. B , Viability of HK-2 cells treated with ginsenoside Rg1 (0, 50, 100, 150, and 200 μM) or pretreated with 5 μg/mL lipopolysaccharide (LPS) ( C ). Data are reported as means±SD (n=3). * P<0.05, ** P <0.01, and *** P<0.001 (ANOVA).

Article Snippet: Human renal tubular epithelial cell line HK-2 was obtained from American Type Culture Collection (ATCC, USA), and was cultured in Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 (DMEM-F12, 3:1, Gibco-BRL, USA) supplemented with 10% fetal bovine serum (FBS, Gibco-BRL) in a humidified 5% CO 2 atmosphere at 37°C.

Techniques:

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Ginsenoside Rg1 protects human renal tubular epithelial cells from lipopolysaccharide-induced apoptosis and inflammation damage

doi: 10.1590/1414-431X20176611

Figure Lengend Snippet: Ginsenoside Rg1 inhibited lipopolysaccharide (LPS)-induced HK-2 cells apoptosis. HK-2 cells were treated with ginsenoside Rg1 (0, 50, 100, 150, and 200 μM) in the absence or presence of 5 μg/mL LPS, and then apoptotic cell rate ( A ), and protein expression levels of Bax, Bcl-2, p53, cleaved-caspase3, and pro-caspase3 were assessed ( B and C ). Data are reported as means±SD (n=3). * P<0.05, ** P<0.01, and *** P<0.001 (ANOVA).

Article Snippet: Human renal tubular epithelial cell line HK-2 was obtained from American Type Culture Collection (ATCC, USA), and was cultured in Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 (DMEM-F12, 3:1, Gibco-BRL, USA) supplemented with 10% fetal bovine serum (FBS, Gibco-BRL) in a humidified 5% CO 2 atmosphere at 37°C.

Techniques: Expressing

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Ginsenoside Rg1 protects human renal tubular epithelial cells from lipopolysaccharide-induced apoptosis and inflammation damage

doi: 10.1590/1414-431X20176611

Figure Lengend Snippet: Ginsenoside Rg1 alleviated lipopolysaccharide (LPS)-induced HK-2 cell migration. HK-2 cells were treated with 150 μM of ginsenoside Rg1 and/or 5 μg/mL of LPS, and then relative migration rate was detected. Data are reported as means±SD (n=3). * P<0.05 (ANOVA).

Article Snippet: Human renal tubular epithelial cell line HK-2 was obtained from American Type Culture Collection (ATCC, USA), and was cultured in Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 (DMEM-F12, 3:1, Gibco-BRL, USA) supplemented with 10% fetal bovine serum (FBS, Gibco-BRL) in a humidified 5% CO 2 atmosphere at 37°C.

Techniques: Migration

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Ginsenoside Rg1 protects human renal tubular epithelial cells from lipopolysaccharide-induced apoptosis and inflammation damage

doi: 10.1590/1414-431X20176611

Figure Lengend Snippet: Ginsenoside Rg1 decreased lipopolysaccharide (LPS)-induced increase in reactive oxygen species (ROS) activity. HK-2 cells were treated with 150 μM of ginsenoside Rg1 and/or 5 μg/mL of LPS, and then ROS activity was detected. Data are reported as means±SD (n=3). ** P<0.01 and *** P<0.001 (ANOVA).

Article Snippet: Human renal tubular epithelial cell line HK-2 was obtained from American Type Culture Collection (ATCC, USA), and was cultured in Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 (DMEM-F12, 3:1, Gibco-BRL, USA) supplemented with 10% fetal bovine serum (FBS, Gibco-BRL) in a humidified 5% CO 2 atmosphere at 37°C.

Techniques: Activity Assay

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Ginsenoside Rg1 protects human renal tubular epithelial cells from lipopolysaccharide-induced apoptosis and inflammation damage

doi: 10.1590/1414-431X20176611

Figure Lengend Snippet: Ginsenoside Rg1 decreased lipopolysaccharide (LPS)-induced inflammation response. HK-2 cells were treated with ginsenoside Rg1 (0, 50, 100, 150, and 200 μM) in the absence or presence of 5 μg/mL LPS, and then the concentrations of ( A ) MCP-1, ( B ) IL-1β, ( C ) IL-6, and ( D ) TNF-α in culture supernatant were measured. Data are reported as means±SD (n=3). * P<0.05, ** P<0.01, and *** P<0.001 (ANOVA).

Article Snippet: Human renal tubular epithelial cell line HK-2 was obtained from American Type Culture Collection (ATCC, USA), and was cultured in Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 (DMEM-F12, 3:1, Gibco-BRL, USA) supplemented with 10% fetal bovine serum (FBS, Gibco-BRL) in a humidified 5% CO 2 atmosphere at 37°C.

Techniques:

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Ginsenoside Rg1 protects human renal tubular epithelial cells from lipopolysaccharide-induced apoptosis and inflammation damage

doi: 10.1590/1414-431X20176611

Figure Lengend Snippet: Ginsenoside Rg1 suppressed lipopolysaccharide (LPS)-induced HK-2 cells injury by activation of PI3K/AKT and inactivation of NF-κB signaling pathways. HK-2 cells were treated with ginsenoside Rg1 (0, 50, 100, 150, and 200 μM) in the absence or presence of 5 μg/mL LPS, and then the protein expressions of p-PI3K, PI3K, p-AKT, AKT, and PTEN, ( A ), as well as the protein expressions of p-IκBα, IκBα, p-p65, p65, and Bcl-3 ( B ) were determined by western blot analysis. C and D , Quantitative analysis based on the results from A and B . Data are reported as means±SD (n=3). * P<0.05, ** P<0.01, and *** P<0.001 (ANOVA).

Article Snippet: Human renal tubular epithelial cell line HK-2 was obtained from American Type Culture Collection (ATCC, USA), and was cultured in Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 (DMEM-F12, 3:1, Gibco-BRL, USA) supplemented with 10% fetal bovine serum (FBS, Gibco-BRL) in a humidified 5% CO 2 atmosphere at 37°C.

Techniques: Activation Assay, Western Blot

Journal: American Journal of Physiology - Renal Physiology

Article Title: Proteinuria causes dysfunctional autophagy in the proximal tubule

doi: 10.1152/ajprenal.00125.2016

Figure Lengend Snippet: Albumin overload inhibits autophagy in cultured primary proximal tubular epithelial cells (PTEC). A: representative immunoblot analysis of LC3 and p62 content in whole cell lysates in primary PTEC treated with increasing concentrations of human recombinant albumin for 5 days. +Bafilomycin, cells treated with 100 nM bafilomycin A1 for 2 h before harvesting and lysis. β-Actin served as a loading control. Quantitative densitometry of p62 expression (n = 3) is also shown. B: autophagic flux calculated using densitometry from Western blots containing LC3-II levels before and after bafilomycin treatment. Values were normalized to β-actin. The change in autophagosome mass, measured by the difference in LC3-II content before and after complete inhibition of fusion for 2 h with bafilomycin is shown. Values are means ± SE; n = 3 independent experiments. C: the magnitude of autophagic flux declines in relation to the concentration of albumin in the apical media. Autophagic flux was determined as shown in Fig. 1B, and individual values were plotted vs. the apical medium albumin concentration. The curve fit was determined by regression analysis, Y = 393.26-255.08log(X), R = 0.89532, n = 12, P < 0.0001. D: the no. of autophagosomes was assessed in live PTEC with monodansylcadaverine (MDC), a fluorescent dye that accumulates in double membrane-bound autophagosomes. Cells were grown in complete primary culture media (control; top) or in EBSS overnight (starvation; bottom) in the absence (left) or presence of albumin (right). MDC-labeled vesicles are induced by starvation, while albumin treatment decreased the no. of MDC-labeled vacuoles. Images were taken with a Nikon Deconvolution Microscope; ×60. Scale bar = 10 μm. Bar graph indicates the average no. of MDC-labeled vesicles/cell ± SD determined by analyzing 20 cells/sample. Three independent experiments were performed obtaining similar results. *P < 0.05 indicates statistical significance compared with control. E: effect of albumin exposure and altered autophagy on the degradation of long-lived proteins. Proteolysis of long-lived proteins was measured in primary mouse PTEC labeled for 48 h in medium containing l-[14C]phenylalanine (Phe) followed by a 48-h chase period in complete or starvation media. After labeling, a 12-h washout period was performed to remove l-[14C]Phe that is released from short-lived proteins. [14C]Phe release was calculated from radioactivity in the tricarboxylic acid-soluble form relative to total cell radioactivity. Data represent 1 of 3 experiments. Values are means ± SE. *P < 0.05, complete media vs. complete media+albumin; starvation media vs. starvation media+albumin.

Article Snippet: HK-2 cells were ordered from ATCC and maintained in DMEM supplemented with 10% FBS, 50 U/ml penicillin, and 50 μg/ml streptomycin.

Techniques: Cell Culture, Western Blot, Recombinant, Lysis, Expressing, Inhibition, Concentration Assay, Labeling, Microscopy, Radioactivity

Journal: American Journal of Physiology - Renal Physiology

Article Title: Proteinuria causes dysfunctional autophagy in the proximal tubule

doi: 10.1152/ajprenal.00125.2016

Figure Lengend Snippet: Proteinuria inhibits LC3-green fluorescent protein (GFP)-labeled autophagosome formation in HK-2 cells. Representative images are shown of direct fluorescence of HK-2 cells transfected with GFP-LC3, an autophagic marker, at baseline (Control), after overnight starvation in EBSS (Starvation), and after albumin treatment for either 2 or 6 days (bottom right); albumin concentration = 5 mg/ml. Images represent random fields from 3 independent experiments. In control cells, abundance of autophagic vesicles was observed, while albumin treatment resulted in a loss of GFP-positive autophagosomes. Bar graph indicates the average no. of GFP-labeled vesicles/cell ± SD determined by analyzing 20 cells/sample. Three independent experiments were performed obtaining similar results. Images were taken with a Nikon Deconvolution Microscope (×60). Scale bar = 10 μm. *P < 0.05, statistically significant compared with control.

Article Snippet: HK-2 cells were ordered from ATCC and maintained in DMEM supplemented with 10% FBS, 50 U/ml penicillin, and 50 μg/ml streptomycin.

Techniques: Labeling, Fluorescence, Transfection, Marker, Concentration Assay, Microscopy