rat renal tubular epithelial cell line nrk52e  (ATCC)

Journal: Aging Cell

Article Title: PAR2‐mediated cellular senescence promotes inflammation and fibrosis in aging and chronic kidney disease

doi: 10.1111/acel.14184

Figure Lengend Snippet: PAR2 activation promotes senescence and enhances chemokine expression in renal epithelial cells. (a) NRK52E renal epithelial cells were subjected to a triple treatment regimen with 150 μM SLIGRL‐NH2 (SLI), administered at 24‐h intervals, for a total duration of 72 h. Control cells were treated with vehicle for 72 h. (b) Representative images of SA‐β‐gal staining with or without PAR2 activation. (c) Quantification of SA‐β‐gal positive cells in cells with or without PAR2 activation. ** p < 0.01 versus control group. (d) Western blots show protein levels of p53 and p21 with or without PAR2 activation. GAPDH was used as internal control. Relative protein expressions were quantified using densitometry. * p < 0.05 versus control group. (e) Representative images of double staining with Cdkn1a ISH staining and followed by SA‐β‐gal staining. (f) Relative mRNA expression of Ccl2 , Ccl7 , Cxcl1 , Il8 , Tnfa , and Il1b in NRK52E cells. * p < 0.01 versus control group. (g) Representative ISH images stained with Ccl2 (red) probe in the cells. (h) Representative pictures of double staining with Ccl2 ISH staining and followed SA‐β‐gal staining. (i) Representative dual ISH staining images of Ccl2 (green) and Cdkn1a (red) gene in the cells.

Article Snippet: NRK52E rat renal tubular epithelial cells were obtained from ATCC (Manassas, VA, USA) and cultured in DMEM supplemented with 5% fetal bovine serum (FBS).

Techniques: Activation Assay, Expressing, Control, Staining, Western Blot, Double Staining

Journal: Aging Cell

Article Title: PAR2‐mediated cellular senescence promotes inflammation and fibrosis in aging and chronic kidney disease

doi: 10.1111/acel.14184

Figure Lengend Snippet: PAR2‐mediated cellular senescence is associated with defective fatty acid oxidation. (a) Cellular triglyceride contents were quantified in NRK52E cells treated with 150 μM of SLIGRL‐NH2 (SLI) or/and 50 μM of oleic acid (OA). * p < 0.05 and *** p < 0.001 versus control group. ### p < 0.001 versus OA‐treated group. (b) Lipid accumulation was visualized by Oil red O staining in NRK52E cells treated with SLI (150 μM) or/and OA (50 μM). (c) Protein levels of PPARα, Acox1, Cpt1α, phosphorylated AMPK, and AMPK were measured using western blotting in SLI‐treated cells. Relative protein expressions were quantified using densitometry. * p < 0.05 versus control group. (d) NRK52E cells were transfected with PPARα and PPRE plasmid for 24 h, followed by treatment with SLIGRL‐NH2. PPARα activity was measured using PPRE luciferase activity. ### p < 0.001 versus PPRE‐transfected group. * p < 0.05 versus PPRE + PPARα‐transfected group. (e) Representative ISH images stained with Cpt1a (red) probe in the cells. (f) The level of lactate in NRK52E cells was quantified with or without PAR2 activation. * p < 0.01 versus control group. (g) Cellular oxygen consumption rates (OCR) were measured using Seahorse systems under PAR2‐activated condition. ** p < 0.01 versus control group. (h) OCR were measured using Seahorse systems under PAR2‐activated condition with or without Rotenone and Antimycin A treatment. # p < 0.05 versus control group. *** p < 0.001 between two groups. (i) Cellular triglyceride contents were quantified in NRK52E cells treated at designated conditions. ** p < 0.01 between two groups. # p < 0.05 versus control group. (j) Representative images of SA‐β‐gal staining under etomoxir‐treated condition. (k) Relative mRNA expression of chemokines ( Ccl2, Ccl7 , and Cxcl1 ) in NRK52E cells treated with or without Etomoxir. * p < 0.01 versus vehicle group (l) * p < 0.01 versus vehicle group. Representative dual ISH images of Ccl2 (green) and Cdkn1a (red) genes under etomoxir‐treated condition.

Article Snippet: NRK52E rat renal tubular epithelial cells were obtained from ATCC (Manassas, VA, USA) and cultured in DMEM supplemented with 5% fetal bovine serum (FBS).

Techniques: Control, Staining, Western Blot, Transfection, Plasmid Preparation, Activity Assay, Luciferase, Activation Assay, Expressing