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