Journal: EBioMedicine

Article Title: PPARγ maintains the metabolic heterogeneity and homeostasis of renal tubules

doi: 10.1016/j.ebiom.2018.10.072

Figure Lengend Snippet: PPARγ suppresses glycolysis via inhibition of EGFR in PT cells. (A) NRK-52E cells with or without PPARγ depletion were transfected with a control vector or a vector expressing PPARγ. Immunoblotting analyses were performed with the indicated antibodies including PPARγ, HK2, PFKL, GLUT, LDHA. All the experiments data were performed in triplicate at least and the representative imagines were shown. (B, C) The media of serum-starved NRK-52E cells with or without PPARγ depletion and with or without reconstituted expression of PPARγ were collected for analysis of glucose consumption (B) and lactate production(C). The data represent the mean ± SD from n = 3 independent experiments. *** P ≤ .001. (D) NRK-52E cells with or without PPARγ depletion were lysed. Immunoblotting analyses were performed with the indicated antibodies including PPARγ, P-EGFR, Total-EGFR, P-ERK1/2, total-ERK1, P-AKT, Total-AKT. All the experiments data were performed in triplicate at least and the representative imagines were shown. (E) NRK-52E cells with or without PPARγ depletion were treated with or without EGFR inhibitor AG1478 (2 μM) for 48 h. Immunoblotting analyses were performed with the indicated antibodies including PPARγ, P-EGFR, HK2, PFKL, GLUT1, LDHA. All the experiments data were performed in triplicate at least and the representative imagines were shown. (F) NRK-52E cells with or without PPARγ overexpression were treated with or without EGF (100 ng/ml) for 24 h. Immunoblotting analyses were performed with the indicated antibodies including PPARγ, HK2, PFKL, GLUT1, GAPDH, LDHA. All the experiments data were performed in triplicate at least and the representative imagines were shown. (G, H) The media of serum-starved NRK-52E cells with or without PPARγ depletion in the presence or absence of AG1478 (2 μM) for 48 h were collected for analysis of glucose consumption (G) and lactate production (H). The data represent the mean ± SD from n = 3 independent experiments. * P ≤ .05;** P ≤ .01; *** P ≤ .001.

Article Snippet: Subsequently, the membrane was washed with 1 × PBS to remove Ponceau S and blocked with 5% ( w / v ) fat-free milk in TBST at room temperature for 1 h. Then the membrane was probed with primary antibodies (anti-PPARα (1:1000 dilution, Abcam),anti-Glut1 (1:1000 dilution, Abcam), anti-NCC (1:1000 dilution, Abcam), anti-iRhom2(1:500 dilution, Abcam), anti-c-Myc(1:1000 dilution, Abcam) anti-CD28K(1:1000 dilution, Cell Signaling Technology) anti-PFKL(1:1000 dilution, Cell Signaling Technology), anti-β-tubulin(1:2000 dilution, Cell Signaling Technology), anti-EGFR(1:2000 dilution, Cell Signaling Technology), anti-phospho-EGFR(1:2000 dilution, Cell Signaling Technology), anti-phospho-ERK(1:2000 dilution, Cell Signaling Technology), anti-phospho-AKT(1:2000 dilution, Cell Signaling Technology), anti-AKT(1:2000 dilution, Cell Signaling Technology), anti-PPARγ(1:1000 dilution, Proteintech), anti-ACOX1(1:1000 dilution, Proteintech), anti-ACSL1(1:1000 dilution, Proteintech), anti-ACAA1(1:1000 dilution, Proteintech), anti-ACAA2(1:1000 dilution, Proteintech), anti-HADH(1:1000 dilution, Proteintech), anti-HK2(1:1000 dilution, Proteintech), anti-LDHA(1:5000 dilution, Proteintech) anti-EGF(1:1000 dilution, Bioworld), anti-CD36(1:1000 dilution, Bioworld), anti-Podocin(1:1000 dilution, Santa Cruz Biotechnology) and anti-ERK1(1:1000 dilution, Santa Cruz Biotechnology)) at 4 °C overnight, The membrane was then washed with 1 × TBST at room temperature 3 times for 5 min and probed with corresponding second antibody at room temperature for 1 h. The final signals of WB fragments were developed by Chemiluminescent Horseradish Peroxidase (HRP) Substrate Reagent (Millipore, Billerica, MA, USA) and then were detected with ChemiDoc™ XRS+ (Bio-Rad, Hercules, CA, USA).

Techniques: Inhibition, Transfection, Plasmid Preparation, Expressing, Western Blot, Over Expression

Journal: EBioMedicine

Article Title: PPARγ maintains the metabolic heterogeneity and homeostasis of renal tubules

doi: 10.1016/j.ebiom.2018.10.072

Figure Lengend Snippet: PPARγ suppresses the glycolysis in PT through iRhom2-mediated downregulation of EGF expression and secretion. (A) Real time PCR analyses of mRNA levels of the indicated gene in NRK-52E cells with or without PPARγ depletion were performed. The data represent the mean ± SD from n = 3 independent experiments. *** P ≤ .0 01. ns represents not significant difference between the indicated sample and the counterpart in the absence of PPARγ depletion. (B) The intracellular and secreted EGF levels of NRK-52E cells with or without PPARγ depletion were analyzed by immunoblotting assay with the indicated antibodies including PPARγ and EGF (left) and ELISA (right), respectively. * P ≤ .05. (C) NRK-52E cells with or without PPARγ depletion were treated with or without EGF neutralizing antibody for 48 h. Immunoblotting analyses were performed with the indicated antibodies including PPARγ, P-EGFR, Total-EGFR. All the experiments data were performed in triplicate at least and the representative imagines were shown. (D) The mRNA levels of PPAR γ and RHBDF2 of NRK-52E cells with or without PPARγ depletion were determined by real time PCR analysis. The data represent the mean ± SD from n = 3 independent experiments. *** P ≤ .001. (E, F) NRK-52E cells with or without PPARγ depletion were transfected with or without a vector expressing iRhom2. Immunoblotting analyses with the indicated antibodies including PPARγ, iRhom2, EGF, P-EGFR, Total-EGFR, P-ERK1/2, Total-ERK1, P-AKT, Total-AKT, HK2, PFKL, GLUT1, LDHA (E) and ELISA analyses of secreted EGF levels (F) were performed. The data represent the mean ± SD from n = 3 independent experiments. ** P ≤ .01. (G) Sequence alignment of the putative PPRE within the human, rat, and mouse RHBDF2 promoters. The varied nucleotides in WT RHBDF2 promoter of different species and mutated nucleotides in the RHBDF2 promoter used for luciferase assay were labeled in red. (H) ChIP analyses of NRK-52E cells were performed with an anti-PPARγ antibody and the primers for the putative PPRE region of RHBDF2 promoter. (I) Luciferase reporter vectors with species-specific or mutated RHBDF2 promoter were co-transfected with the vector expressing PPARγ into HEK293FT cells treated with or without PPARγ ligand Rosiglitazone (TZD) (50 μM). Luciferase reporter analyses were performed. The data represent the mean ± SD from n = 3 independent experiments. ** P ≤ .01; *** P ≤ .001. (J, K) Serum-starved NRK52-E cells with or without PPARγ depletion or iRhom2 overexpression were treated with or without EGF (100 ng/ml) for 24 h. The media were collected for analysis of glucose consumption (j) and lactate production (k). The data represent the mean ± SD from n = 3 independent experiments. * P ≤ .05; ** P ≤ .01.

Article Snippet: Subsequently, the membrane was washed with 1 × PBS to remove Ponceau S and blocked with 5% ( w / v ) fat-free milk in TBST at room temperature for 1 h. Then the membrane was probed with primary antibodies (anti-PPARα (1:1000 dilution, Abcam),anti-Glut1 (1:1000 dilution, Abcam), anti-NCC (1:1000 dilution, Abcam), anti-iRhom2(1:500 dilution, Abcam), anti-c-Myc(1:1000 dilution, Abcam) anti-CD28K(1:1000 dilution, Cell Signaling Technology) anti-PFKL(1:1000 dilution, Cell Signaling Technology), anti-β-tubulin(1:2000 dilution, Cell Signaling Technology), anti-EGFR(1:2000 dilution, Cell Signaling Technology), anti-phospho-EGFR(1:2000 dilution, Cell Signaling Technology), anti-phospho-ERK(1:2000 dilution, Cell Signaling Technology), anti-phospho-AKT(1:2000 dilution, Cell Signaling Technology), anti-AKT(1:2000 dilution, Cell Signaling Technology), anti-PPARγ(1:1000 dilution, Proteintech), anti-ACOX1(1:1000 dilution, Proteintech), anti-ACSL1(1:1000 dilution, Proteintech), anti-ACAA1(1:1000 dilution, Proteintech), anti-ACAA2(1:1000 dilution, Proteintech), anti-HADH(1:1000 dilution, Proteintech), anti-HK2(1:1000 dilution, Proteintech), anti-LDHA(1:5000 dilution, Proteintech) anti-EGF(1:1000 dilution, Bioworld), anti-CD36(1:1000 dilution, Bioworld), anti-Podocin(1:1000 dilution, Santa Cruz Biotechnology) and anti-ERK1(1:1000 dilution, Santa Cruz Biotechnology)) at 4 °C overnight, The membrane was then washed with 1 × TBST at room temperature 3 times for 5 min and probed with corresponding second antibody at room temperature for 1 h. The final signals of WB fragments were developed by Chemiluminescent Horseradish Peroxidase (HRP) Substrate Reagent (Millipore, Billerica, MA, USA) and then were detected with ChemiDoc™ XRS+ (Bio-Rad, Hercules, CA, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Transfection, Plasmid Preparation, Sequencing, Luciferase, Labeling, Over Expression