Journal: Anatomy & Cell Biology

Article Title: Cyclosporin A aggravates hydrogen peroxide-induced cell death in kidney proximal tubule epithelial cells

doi: 10.5115/acb.18.192

Figure Lengend Snippet: Cyclosporin A (CsA) enhances hydrogen peroxide (H 2 O 2 ) injury in human kidney proximal tubule epithelial cells. Human kidney proximal tubule epithelial HK-2 cells were cultured in RPMI 1640 until reaching 80% confluence. (A) HK-2 cells were treated with either CsA (1, 10, or 100 nM) or 1% dimethyl sulfoxide (vehicle) for 1 hour and then exposed to 1 mM H 2 O 2 or distilled water (control) for 60 minutes. Cell viability was measured using MTT assay (n=9 wells from 3 experiments per condition). In box plots, whiskers represent the minimum and maximum; bases represent the interquartile range between the first and third quartiles; and midlines represent the median. (B) HK-2 cells were treated with either 10 nM CsA or vehicle for 60 minutes and then exposed to 1 mM H 2 O 2 or control for 30, 60, or 120 minutes. Cell viability was measured using MTT assay (n=9 wells from 3 experiments per condition). (C, D) HK-2 cells were treated with either 10 nM CsA or vehicle for 60 minutes and then exposed to 1 mM H 2 O 2 or control for 60 minutes. Cell death was analyzed by flow cytometry with an annexin V-FITC detection kit after treatment with FITC-conjugated annexin V and propidium iodide. The flow cytometry assay distinguishes among normal (lower left), early apoptosis (lower right), late apoptosis (upper right), and necrosis (upper left). Three experiments were performed to evaluate the cell death. In each experiment, three samples per experimental condition were included. a) P <0.05 vs. control. b) P <0.05 vs. vehicle. c) P <0.01 vs. 0 mol/l.

Article Snippet: HK-2 human kidney proximal tubule epithelial cell line as purchased from the American Type Culture Collection (Rockville, MD, USA) was cultured in RPMI 1640 medium (Welgene, Daegu, Korea), supplemented with 10% fetal bovine serum (Welgene) at 37°C with 5% CO 2 , as described previously [ ].

Techniques: Cell Culture, Control, MTT Assay, Flow Cytometry

Journal: Anatomy & Cell Biology

Article Title: Cyclosporin A aggravates hydrogen peroxide-induced cell death in kidney proximal tubule epithelial cells

doi: 10.5115/acb.18.192

Figure Lengend Snippet: Cyclosporin A (CsA) increases p53 activation and BH3 interacting-domain death agonist (BID) expression after H 2 O 2 injury in human kidney proximal tubule epithelial cells. HK-2 cells were treated with either 10 nM CsA or vehicle for 60 minutes and then exposed to 1 mM H 2 O 2 or control for 60 minutes (n=6 experiments per condition). (A) Phosphorylation level of p53 (p-p53, 53 kDa) and total expression level of p53 (t-p53, 53 kDa) were measured by western blot analysis. Antibody of β-actin (43 kDa) as a loading control was used for normalization. (B–D) Intensities of p-p53 and t-p53 protein bands were quantified using the AzureSpot software. (E) BID (22 kDa) expression was measured by western blot analysis. Antibody of β-actin (43 kDa) as a loading control was used for normalization. (F) Intensity of BID protein expression was quantified using the AzureSpot software. a) P <0.05 vs. control. b) P <0.05 vs. vehicle.

Article Snippet: HK-2 human kidney proximal tubule epithelial cell line as purchased from the American Type Culture Collection (Rockville, MD, USA) was cultured in RPMI 1640 medium (Welgene, Daegu, Korea), supplemented with 10% fetal bovine serum (Welgene) at 37°C with 5% CO 2 , as described previously [ ].

Techniques: Activation Assay, Expressing, Control, Phospho-proteomics, Western Blot, Software

Journal: Anatomy & Cell Biology

Article Title: Cyclosporin A aggravates hydrogen peroxide-induced cell death in kidney proximal tubule epithelial cells

doi: 10.5115/acb.18.192

Figure Lengend Snippet: Cyclosporin A (CsA) does not alter activations of mitogen-activated protein kinases and protein kinase B (AKT) after H 2 O 2 injury in human kidney proximal tubule epithelial cells. HK-2 cells were treated with either 10 nM CsA or vehicle for 60 minutes and then exposed to 1 mM H 2 O 2 or control for 60 minutes (n=6 experiments per condition). (A) Phosphorylation levels of p38 (p-p38, 43 kDa), c-Jun N-terminal kinase (p-JNK, 46 and 55 kDa), and extracellular signal-regulated kinase (p-ERK, 42 and 44 kDa), and their total expression levels (t-p38, p-JNK, and p-ERK) were measured by western blot analysis. Antibody of β-actin (43 kDa) as a loading control was used for normalization. (B, C, E, F, H, I) Intensities of p-p38, t-p38, p-JNK, total JNK (t-JNK), p-ERK, and total ERK (t-ERK) protein bands were quantified using the AzureSpot software. (D, G, J) Activations of p38, JNK, and ERK indicated by their respective ratio of phosphorylation level to total expression level. (K) AKT phosphorylation (p-AKT) and total expression (t-AKT) with a molecular mass of 56 to 62 kDa were measured by western blot analysis. Antibody of β-actin (43 kDa) as a loading control was used for normalization. (L, M) Intensities of p-AKT and t-AKT protein bands were quantified using the AzureSpot software. (N) AKT activation based on the ratio of phosphorylation to total expression. a) P <0.05 vs. control.

Article Snippet: HK-2 human kidney proximal tubule epithelial cell line as purchased from the American Type Culture Collection (Rockville, MD, USA) was cultured in RPMI 1640 medium (Welgene, Daegu, Korea), supplemented with 10% fetal bovine serum (Welgene) at 37°C with 5% CO 2 , as described previously [ ].

Techniques: Control, Phospho-proteomics, Expressing, Western Blot, Software, Activation Assay

Journal: Anatomy & Cell Biology

Article Title: Cyclosporin A aggravates hydrogen peroxide-induced cell death in kidney proximal tubule epithelial cells

doi: 10.5115/acb.18.192

Figure Lengend Snippet: Cyclosporin A (CsA) enhances reactive oxygen species (ROS) production induced by H 2 O 2 in human kidney proximal tubule epithelial cells without altering expression level of antioxidant enzymes. HK-2 cells were treated with either 10 nM CsA or vehicle for 60 minutes and then exposed to 1 mM H 2 O 2 or control for 60 minutes (n=3 experiments per condition). (A) ROS production was measured using oxidative sensitive dye 2′,7′-dichlorodihydrofluorescein diacetate. (B) Expression levels of manganese superoxide dismutase (MnSOD; 25 kDa), copper/zinc superoxide dismutase (CuZnSOD; 23 kDa), and catalase (64 kDa) were measured by western blot analysis. Antibody of β-actin (43 kDa) as a loading control was used for normalization. (C–E) Intensities of MnSOD, CuZnSOD, and catalase protein bands were quantified using the AzureSpot software. N.S., not significant. * P <0.05 vs. control, # P <0.05 vs. vehicle.

Article Snippet: HK-2 human kidney proximal tubule epithelial cell line as purchased from the American Type Culture Collection (Rockville, MD, USA) was cultured in RPMI 1640 medium (Welgene, Daegu, Korea), supplemented with 10% fetal bovine serum (Welgene) at 37°C with 5% CO 2 , as described previously [ ].

Techniques: Expressing, Control, Western Blot, Software

Journal: Anatomy & Cell Biology

Article Title: Cyclosporin A aggravates hydrogen peroxide-induced cell death in kidney proximal tubule epithelial cells

doi: 10.5115/acb.18.192

Figure Lengend Snippet: Cyclosporin A (CsA) increases mitochondrial depolarization induced by H 2 O 2 in human kidney proximal tubule epithelial cells. HK-2 cells were treated with either 10 nM CsA or vehicle for 60 minutes and then exposed to 1 mM H 2 O 2 or control for 60 minutes. (A) Percentage of mitochondrial membrane potential was measured using tetramethylrhodamine ethyl ester perchlorate dye (n=9 wells per 3 experiments per condition). The mitochondrial membrane potential in the group with control plus vehicle was taken as 100%. a) P <0.05 vs. control. b) P <0.05 vs. vehicle. (B) Expression levels of 94 kDa glucose-regulated protein (GRP94) and 78 kDa glucose-regulated protein (GRP78) were measured by western blot analysis. Antibody of β-actin (43 kDa) as a loading control was used for normalization. (C, D) Intensities of GRP94 and GRP78 protein bands were quantified using the AzureSpot software (n=6 experiments per condition). N.S., not significant.

Article Snippet: HK-2 human kidney proximal tubule epithelial cell line as purchased from the American Type Culture Collection (Rockville, MD, USA) was cultured in RPMI 1640 medium (Welgene, Daegu, Korea), supplemented with 10% fetal bovine serum (Welgene) at 37°C with 5% CO 2 , as described previously [ ].

Techniques: Control, Membrane, Expressing, Western Blot, Software