Journal: eLife

Article Title: The spatial separation of processing and transport functions to the interior and periphery of the Golgi stack

doi: 10.7554/eLife.41301

Figure Lengend Snippet:

Article Snippet: Cell line ( Rattus norvegicus ) , Normal rat kidney (NRK) fibroblast cell , ATCC , ATCC: CRL-1570; RRID: CVCL_2144 , .

Techniques: Recombinant, Plasmid Preparation, Antibody Labeling, Software

Journal: Journal of Cellular and Molecular Medicine

Article Title: Conditioned mesenchymal stem cells attenuate progression of chronic kidney disease through inhibition of epithelial-to-mesenchymal transition and immune modulation

doi: 10.1111/j.1582-4934.2012.01610.x

Figure Lengend Snippet: Effects of conditioned MSCs on expression of α-SMA and fibronectin in renal interstitial fibroblast NRK-49F. NRK-49F cells were cultured without or with 15 ng/ml TGF-β1 alone or co-culture with conditioned MSCs and/or ascorbic acid 2- phosphate for 3 days. ( A ) Representative Western blot analysis and relative bar graph analysis for α-SMA and β-tubulin level. ( B ) Representative Western blot and relative bar graph analysis of fibronectin protein level in NRK-49F after various treatments. * P < 0.05 versus normal control; ** P < 0.05 versus TGF-β1 treated.

Article Snippet: Rat renal proximal tubular cells (NRK-52E) and normal rat kidney interstitial fibroblast cells (NRK-49F) were purchased from the Bioresource Collection and Research Center of the Food Industry Research Institute, Taiwan.

Techniques: Expressing, Cell Culture, Co-Culture Assay, Western Blot, Control

Journal: BioMed Research International

Article Title: Deletion of Akt1 Promotes Kidney Fibrosis in a Murine Model of Unilateral Ureteral Obstruction

doi: 10.1155/2020/6143542

Figure Lengend Snippet: Akt1 deletion is associated with upregulation of TGF β 1. (a) Immunohistochemical staining (×200). Expression of TGF β 1 began to increase from day 1 after UUO in Akt1 −/− mice. TGF β 1 was expressed more strongly in Akt1 −/− mice than in wild type mice as UUO progressed. (b) Western blot analysis. TGF β 1 expression was higher in Akt1 −/− mice than in wild type mice on day 0 (U0 vs. A0, P = 0.0022) and day 1 (U1 vs. A1, P = 0.0260) but was not statistically different between the two groups on day 3 (U3 vs. A3, P = 0.0931) and day 7 (U7 vs. A7, P = 0.1797). (c) Western blot analysis. In HK2 cells, silencing of Akt1 promoted the expression of TGF β 1 and fibronectin in the unstimulated state, which was augmented by angiotensin II stimulation (1 μ M). (d) Western blot analysis. In NRK-49F cells, silencing of Akt1 did not affect the expression of TGF β 1 and fibronectin regardless of angiotensin II stimulation. Abbreviations: ANGII: angiotensin II; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; HK2 cells: immortalized human proximal tubular cells; NRK-49F cells: rat kidney fibroblasts; TGF β 1: transforming growth factor β 1; UUO: unilateral ureteral obstruction; A0 ( n = 6): Akt1 −/− sham; A1 ( n = 6): day 1 Akt1 −/− ; A3 ( n = 6): day 3 Akt1 −/− ; A7 ( n = 6): day 7 Akt1 −/− ; U0 ( n = 6): wild type sham; U1 ( n = 6): day 1 wild type; U3 ( n = 6): day 3 wild type; U7 ( n = 6): day 7 wild type.

Article Snippet: Rat kidney fibroblasts (NRK-49F cells) were obtained from ATCC (Manassas, VA, USA) and cultured in DMEM containing 4.5 g/L D-glucose, 5% fetal bovine serum, and penicillin-streptomycin.

Techniques: Immunohistochemical staining, Staining, Expressing, Western Blot

Journal: BioMed Research International

Article Title: Deletion of Akt1 Promotes Kidney Fibrosis in a Murine Model of Unilateral Ureteral Obstruction

doi: 10.1155/2020/6143542

Figure Lengend Snippet: Akt1 deletion is associated with activation of STAT3 independently of the Smad pathway. (a) Western blot analysis. In NRK-49F cells, there were no differences in the expression of phosphorylated- (p-) Smad2/3 and Smad7 both in pLKO and shAkt1 groups irrespective of TGF β 1 treatment (10 ng/mL). (b) Western blot analysis. In NRK-49F and HK2 cells, p-STAT3/STAT3 ratios were significantly elevated in the pLKO group treated with TGF β 1 and the shAkt1 group compared with the pLKO group not treated with TGF β ( ∗ P < 0.05). (c) Immunohistochemical staining. After UUO, the expression of p-STAT3 was more increased in Akt1 −/− mice compared to wild type mice even from day 0 (×200). Picture surrounded by a black box shows the pattern of expression of p-STAT3 in tubular cells and surrounding cells in day 7 Akt1 −/− kidney (×400, black arrows: brown color-stained cells). Abbreviations: GAPDH: glyceraldehyde 3-phosphate dehydrogenase; NRK-49F cells: rat kidney fibroblasts; STAT3: signal transducer and activator of transcript 3; TGF β 1: transforming growth factor β 1; UUO: unilateral ureteral obstruction; A0 ( n = 6): Akt1 −/− sham; A1 ( n = 6): day 1 Akt1 −/− ; A3 ( n = 6): day 3 Akt1 −/− ; A7 ( n = 6): day 7 Akt1 −/− ; U0 ( n = 6): wild type sham; U1 ( n = 6): day 1 wild type; U3 ( n = 6): day 3 wild type; U7 ( n = 6): day 7 wild type.

Article Snippet: Rat kidney fibroblasts (NRK-49F cells) were obtained from ATCC (Manassas, VA, USA) and cultured in DMEM containing 4.5 g/L D-glucose, 5% fetal bovine serum, and penicillin-streptomycin.

Techniques: Activation Assay, Western Blot, Expressing, Immunohistochemical staining, Staining

Journal: BioMed Research International

Article Title: Deletion of Akt1 Promotes Kidney Fibrosis in a Murine Model of Unilateral Ureteral Obstruction

doi: 10.1155/2020/6143542

Figure Lengend Snippet: Akt1 deletion promotes apoptosis. (a) Immunohistochemical staining. After UUO, cleaved- (c-) caspase-3 was expressed strongly in the nuclei of the renal tubular epithelial on day 7 in both Akt1 −/− and wild type mice (black arrow) (×400). (b) Quantification of stained cells for c-caspase-3. Expression of c-caspase-3 was significantly higher in Akt1 −/− mice than in wild type mice on day 7. (c) Western blot analysis. In HK2 cells, knockdown of Akt1 led to increased expression of c-caspase-3 in the unstimulated state, which remained elevated upon TGF β 1 stimulation (10 ng/mL). Bax also increased its expression by TGF β 1 stimulation in the shAkt1 group with cleaved caspase-3. In NRK-49F cells, knockdown of Akt1 did not affect the expression of c-caspase-3 regardless of TGF β 1 stimulation. Abbreviations: HK2 cells: immortalized human proximal tubular cells; NRK-49F cells: rat kidney fibroblasts; TGF β 1: transforming growth factor β 1; UUO: unilateral ureteral obstruction; A0 ( n = 6): Akt1 −/− sham; A1 ( n = 6): day 1 Akt1 −/− ; A3 ( n = 6): day 3 Akt1 −/− ; A7 ( n = 6): day 7 Akt1 −/− ; U0 ( n = 6): wild type sham; U1 ( n = 6): day 1 wild type; U3 ( n = 6): day 3 wild type; U7 ( n = 6): day 7 wild type. ∗ P < 0.05.

Article Snippet: Rat kidney fibroblasts (NRK-49F cells) were obtained from ATCC (Manassas, VA, USA) and cultured in DMEM containing 4.5 g/L D-glucose, 5% fetal bovine serum, and penicillin-streptomycin.

Techniques: Immunohistochemical staining, Staining, Expressing, Western Blot, Knockdown

Journal: The American Journal of Pathology

Article Title: Activation of AMP-Activated Protein Kinase Prevents TGF-β1–Induced Epithelial-Mesenchymal Transition and Myofibroblast Activation

doi: 10.1016/j.ajpath.2015.04.014

Figure Lengend Snippet: Primer Sequences Used to Analyze mRNA Expression in Mouse Kidney Cortex and Normal Rat Kidney Interstitial Fibroblast Cells

Article Snippet: Normal rat kidney interstitial fibroblast cells (NRK-49F) were obtained from ATCC (Manassas, VA) grown in RPMI low-glucose medium supplemented with 10% FBS.

Techniques: Expressing

Journal: The American Journal of Pathology

Article Title: Activation of AMP-Activated Protein Kinase Prevents TGF-β1–Induced Epithelial-Mesenchymal Transition and Myofibroblast Activation

doi: 10.1016/j.ajpath.2015.04.014

Figure Lengend Snippet: AMP-activated protein kinase (AMPK) inhibits transforming growth factor (TGF)-β1–induced matrix accumulation in normal rat kidney interstitial fibroblasts. A: Quiescent cells were stimulated with 1 ng/mL of TGF-β1 for the indicated periods. Cell lysates were immunoblotted with fibronectin, α-smooth muscle actin (α-SMA), p-AMPKα (Thr172), and AMPKα antibodies. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Blots are representative of three independent experiments. B: Quiescent cells were pretreated with 0.5 mmol/L adenine 9-β-d-arabinofuranoside (ARA) for 30 minutes followed by 1-ng/mL TGF-β1 treatment for 24 hours. Cell lysates were immunoblotted with fibronectin, α-SMA, p-AMPKα (Thr172), and AMPKα antibodies. GAPDH was used as a loading control (left panel). Cells were infected with adenovirus encoding dominant negative AMPK (Ad-DN-AMPK) at a multiplicity of infection of 50 for 24 hours. The green fluorescent protein vector (Ad-GFP) was used as experimental control. Cells were serum starved for 16 hours before 1-ng/mL TGF-β1 treatment. Cell lysates were immunoblotted with fibronectin, α-SMA, AMPK, and GAPDH antibodies (right panel). Blots are representative of two independent experiments. C: Quiescent cells were pretreated with 1 mmol/L 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR) or 1 mmol/L metformin for 30 minutes followed by 1-ng/mL TGF-β1 treatment for 24 hours. Cell lysates were immunoblotted with fibronectin, α-SMA, p-AMPKα (Thr172), and AMPKα antibodies. GAPDH was used as a loading control. Blots are representative of three independent experiments. D: Quiescent cells were pretreated with 1 mmol/L AICAR for 30 minutes followed by 1-ng/mL TGF-β1 treatment for 24 hours. Cells were immunostained with fibronectin (green) and α-SMA (red) antibodies. Nuclei were stained with DAPI (blue). E: Fibronectin and α-SMA mRNAs were measured by quantitative RT-PCR on RNA extracted from cells treated with TGF-β1 in the presence or absence of ARA or AICAR. ∗∗∗P < 0.001 versus control by analysis of variance; ††P < 0.01, †††P < 0.001 versus TGF-β1 by analysis of variance.

Article Snippet: Normal rat kidney interstitial fibroblast cells (NRK-49F) were obtained from ATCC (Manassas, VA) grown in RPMI low-glucose medium supplemented with 10% FBS.

Techniques: Control, Infection, Dominant Negative Mutation, Plasmid Preparation, Staining, Quantitative RT-PCR