human primary renal proximal tubular epithelial cells (ptecs)  (Lonza)

Journal: International Journal of Molecular Sciences

Article Title: Inhibition of BRD4 Reduces Neutrophil Activation and Adhesion to the Vascular Endothelium Following Ischemia Reperfusion Injury

doi: 10.3390/ijms21249620

Figure Lengend Snippet: BRD4 attenuated the gene expression of NFκB-mediated genes. ( A – C ) Exposure time was optimized by treating PTECs with H 2 O 2 for 0, 1, 6, and 24 h before cells were collected to qPCR analysis of IL6 ( A ), CXCL2 ( B ) and CCL2 ( C ). ( D – F ) In a subsequent set of studies, cells were treated with 1 μM MS417 for 2 h followed by H 2 O 2 for 6 h followed by qPCR. n = 3, * p < 0.05, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human primary renal proximal tubular epithelial cells (PTECs) were purchased from Lonza, Basel, Switzerland.

Techniques: Gene Expression

Journal: bioRxiv

Article Title: Proteomics Reveals Extracellular Matrix Injury in the Glomeruli and Tubulointerstitium of Kidney Allografts with Early Antibody-Mediated Rejection

doi: 10.1101/2020.03.03.975672

Figure Lengend Snippet: Validation of proteomic findings in the AMR tubulointerstitium in primary human proximal tubular epithelial cells Protein expression of GSTO1 was analyzed in PTECs after treatment with vehicle, 20ng/µL of TNFα, 1000U/mL of IFNγ, or 1ng/µL of LPS for 24h, and normalized to GAPDH (A). The immunoblots and corresponding densitometry values are shown. GSTO1 expression was also measured at the gene level after treatment with vehicle, 20ng/µL of TNFα, or 1000U/mL of IFNγ for 24h (A). The effects of TNFα and IFNγ on the gene expression of CTSL, CTSV, CTSS, and LGMN proteases, were studied (B). OCR and ECAR were monitored in a Seahorse XFe96 analyzer in order to study the effects of TNFα, IFNγ, or LPS on PTECs. As expected, LPS-treated PTECs (positive control) experienced a metabolic switch towards a more glycolytic phenotype, compared to vehicle-treated cells (C). The following concentrations were employed: oligomycin: 1µM; FCCP: 0.3µM, 2-DG: 100mM; Rot: 1mM; AA: 1mM. Oligomycin induces an increase in glycolysis by inhibiting ATP synthase. FCCP induces mitochondrial stress by uncoupling respiration from ATP synthesis. Rot/AA are electron transport chain inhibitors. *P<0.05 vs. Vehicle-treated PTECs. Intracellular levels of superoxide ion (D) and ATP (E) were also measured in vehicle-, TNFα-, and IFNγ-treated PTECs. Data are expressed as mean ± SEM. *P<0.05; **P<0.01; ***P<0.001. PTECs, human proximal tubular epithelial cells; TNFα, tumor necrosis factor alpha ; IFNγ, interferon gamma; LPS, lipopolysaccharide; GSTO1, glutathione S-transferase omega-1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CTSL, cathepsin-L; CTSV, cathepsin-V; CTSS, cathepsin-S; LGMN, legumain; ACTB, beta-actin; ECAR, extracellular acidification rate; OCR, oxygen consumption rate; FCCP, p-trifluoromethoxy carbonyl cyanide phenyl hydrazone; 2-DG, 2-deoxyglucose; Rot, rotenone; AA: antimycin A.

Article Snippet: Primary human proximal tubular epithelial cells (PTECs, Lonza) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 5.55mM D-glucose, 4mM L-glutamine, and 1mM sodium pyruvate, and supplemented with 10% v/v dialyzed fetal bovine serum (FBS), 10 ng/ml EGF, 1x of Transferrin/Insulin/Selenium (Invitrogen), 0.05M hydrocortisone, 50 units/ml penicillin, and 50g/ml streptomycin, as previously described.

Techniques: Expressing, Western Blot, Positive Control

Journal: Journal of Clinical Medicine

Article Title: The Interaction of miR-378i-Skp2 Regulates Cell Senescence in Diabetic Nephropathy

doi: 10.3390/jcm7120468

Figure Lengend Snippet: High glucose (HG) induces cell senescence in proximal tubular epithelial cells (PTECs). ( A ) The effect of HG on morphological changes and senescence-associated β-galactosidase (SAβ-Gal) staining of human PTECs. PTECs were incubated under normal glucose (NG, 6.2 mM) and HG (30 mM) conditions for seven days and nine days. Cell senescence was assessed using SAβ-Gal staining. HG increased p53 ( B ) (D3: n = 6, D4: n = 4) and p27 ( C ) (D3: n = 3, D4: n = 3), and decreased cyclin E1 ( D ) (D3: n = 3, D4: n = 4) and cyclin E2 ( E ) (D3: n = 3, D4: n = 4) protein expression in human PTECs after three and four days of treatment. Protein levels were assessed by western blot. The expression of p53 in the proximal tubule of kidneys of mice ( F ) and humans ( G ). The kidney sections of C57BL/6 mice, non-diabetic db/m mice, and diabetic db/db mice, and human donors (upper tract urothelial carcinoma, UTUC with normal kidney function and normal glomerulus and proximal tubule) and patients with diabetic nephropathy (DN) were stained with p53 (brown). The images quantification was performed using the IHC Profiler Plugin of ImageJ Software. The bar graph represents the mean ± S.E.M. * p < 0.05, ** p < 0.01, *** p < 0.001 by Student’s t test.

Article Snippet: Primary proximal tubular epithelial cells (PTECs) of a DM patient and a normal individual (Lonza Walkersville Inc., Walkersville, MD, USA) were cultured in Clonetics TM REGM TM BulletKit TM (CC-3190).

Techniques: Staining, Incubation, Expressing, Western Blot, Software

Journal: Journal of Clinical Medicine

Article Title: The Interaction of miR-378i-Skp2 Regulates Cell Senescence in Diabetic Nephropathy

doi: 10.3390/jcm7120468

Figure Lengend Snippet: Identification of potential genes associated with cell senescence in PTECs in DN. ( A ) Flowchart of identification of potential genes associated with cell senescence in PTECs. ( B ) Display of differential expression patterns of normal and diabetic PTECs from deep RNA sequencing by volcano plot. ( C ) The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of differentially expressed genes in DAVID database. The 612 differentially expressed genes in diabetic PTECs were uploaded into DAVID database for enrichment analysis. The top seven KEGG pathway analysis results of these dysregulated genes in diabetic PTECs are displayed in a pie chart. The pie chart indicates the-Log10 (false discovery rate, FDR) of each KEGG term, and the numbers that are shown at the outside of each pie segment indicates the number of genes involved in each term. ( D ) The protein-protein interaction network analysis of 14 genes associated cell cycle of KEGG pathway using STRING database. S-phase kinase protein 2 (Skp2) correlated with cell cycle markers, such as cyclin D2 (CCND2), cyclin B1 (CCNB1), cyclin-dependent kinase inhibitor 1C (CDKN1C), and CDKN2C. ( E ) The potential network of Skp2 mediating cell cycle in Core analysis of Ingenuity Pathway Analysis (IPA) software. Skp2 correlated with cyclins.

Article Snippet: Primary proximal tubular epithelial cells (PTECs) of a DM patient and a normal individual (Lonza Walkersville Inc., Walkersville, MD, USA) were cultured in Clonetics TM REGM TM BulletKit TM (CC-3190).

Techniques: Quantitative Proteomics, RNA Sequencing, Software

Journal: Journal of Clinical Medicine

Article Title: The Interaction of miR-378i-Skp2 Regulates Cell Senescence in Diabetic Nephropathy

doi: 10.3390/jcm7120468

Figure Lengend Snippet: The networks associated with genes differentially expressed in diabetic PTECs in IPA database.

Article Snippet: Primary proximal tubular epithelial cells (PTECs) of a DM patient and a normal individual (Lonza Walkersville Inc., Walkersville, MD, USA) were cultured in Clonetics TM REGM TM BulletKit TM (CC-3190).

Techniques: Histone Deacetylase Assay, Cell Function Assay

Journal: Journal of Clinical Medicine

Article Title: The Interaction of miR-378i-Skp2 Regulates Cell Senescence in Diabetic Nephropathy

doi: 10.3390/jcm7120468

Figure Lengend Snippet: Decreased Skp2 expression is associated with cell senescence of PTECs in DN. ( A ) Decreased Skp2 mRNA expression was found in PTECs from a type 2 diabetes mellitus (DM) patient ( n = 3). ( B ) HG decreased Skp2 mRNA expression in human PTECs after three days of treatment ( n = 4). Skp2 mRNA levels were assessed by quantitative real-time polymerase chain reaction (PCR). ( C ) HG suppressed Skp2 protein expression in human PTECs after three and four days of treatment (D3: n = 3, D4: n = 3). Skp2 protein levels were assessed by western blot. The expression of Skp2 in the proximal tubule of kidneys of mice ( D ) and humans ( E ). The kidney sections of C57BL/6 mice, non-diabetic db/m mice, and diabetic db/db mice, and human donors (UTUC with normal kidney function and normal glomerulus and proximal tubule) and patients with DN were stained with Skp2 (brown). The images quantification was performed using the IHC Profiler Plugin of ImageJ Software. The bar graph represents the mean ± S.E.M. * p < 0.05, *** p < 0.001 by Student’s t test.

Article Snippet: Primary proximal tubular epithelial cells (PTECs) of a DM patient and a normal individual (Lonza Walkersville Inc., Walkersville, MD, USA) were cultured in Clonetics TM REGM TM BulletKit TM (CC-3190).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Staining, Software

Journal: Journal of Clinical Medicine

Article Title: The Interaction of miR-378i-Skp2 Regulates Cell Senescence in Diabetic Nephropathy

doi: 10.3390/jcm7120468

Figure Lengend Snippet: Identification of miR-378i-Skp2 interaction in cell senescence in diabetic PTECs. ( A ) The heat map revealed differentially expressed miRNAs from normal and diabetic PTECs with Z-score values. ( B ) The regulation of miR-378i on cell cycle, as predicted by IPA. ( C ) Increased miR-378i levels were found in PTECs from a type 2 DM patient ( n = 3). ( D ) HG increased miR-378i levels in human PTECs after three days of treatment ( n = 3). miR-378i levels were assessed by quantitative real-time PCR. ( E ) A schematic representation of sequence alignment of miR-378i and Skp2 mRNA 3’UTR. ( F ) miR-378i mimic suppressed Skp2 expression in HEK 293 cells. Cells were transfected with either control mimic or miR-378i mimic (100 nM) using DharmaFECT No. 1 Transfection Reagent. After 72 h transfection, western blot was utilized to measure Skp2 protein expression. The bar graph represents the mean ± S.E.M. ** p < 0.01 by Student’s t test.

Article Snippet: Primary proximal tubular epithelial cells (PTECs) of a DM patient and a normal individual (Lonza Walkersville Inc., Walkersville, MD, USA) were cultured in Clonetics TM REGM TM BulletKit TM (CC-3190).

Techniques: Real-time Polymerase Chain Reaction, Sequencing, Expressing, Transfection, Control, Western Blot

Journal: Journal of Clinical Medicine

Article Title: The Interaction of miR-378i-Skp2 Regulates Cell Senescence in Diabetic Nephropathy

doi: 10.3390/jcm7120468

Figure Lengend Snippet: Potential microRNA–mRNA interactions identified in diabetic PTECs.

Article Snippet: Primary proximal tubular epithelial cells (PTECs) of a DM patient and a normal individual (Lonza Walkersville Inc., Walkersville, MD, USA) were cultured in Clonetics TM REGM TM BulletKit TM (CC-3190).

Techniques:

Journal: Journal of Clinical Medicine

Article Title: The Interaction of miR-378i-Skp2 Regulates Cell Senescence in Diabetic Nephropathy

doi: 10.3390/jcm7120468

Figure Lengend Snippet: Illustration of the interaction between miR-378i and Skp2 inducing cell senescence in PTECs in DN.

Article Snippet: Primary proximal tubular epithelial cells (PTECs) of a DM patient and a normal individual (Lonza Walkersville Inc., Walkersville, MD, USA) were cultured in Clonetics TM REGM TM BulletKit TM (CC-3190).

Techniques: