mouse podocyte primary cell culture complete medium with serum  (Celprogen Inc)

Journal: International Journal of Biological Sciences

Article Title: Gastrodin alleviates high fructose-induced podocyte mitochondria-mediated apoptosis by inhibiting NLRP6 to facilitate TRIM7-triggered Bok mRNA degradation

doi: 10.7150/ijbs.120307

Figure Lengend Snippet: NLRP6 inhibition attenuates mitochondria-mediated apoptosis under high fructose stimulation. (A) The principal component analysis of WT and Nlrp6 -/- mouse glomeruli with or without HFrD, ( n = 6). (B) Venn diagram shows the overlapped unique differentially expressed genes between WT-HFrD versus WT-NC and Nlrp6 -/- -HFrD versus WT-HFrD from the data of RNA-seq analysis of mouse-isolated glomeruli. (C-E) Heatmap shows the overlapped gene (downregulation), and analyzed by KEGG and GO. (F) Western blot detection of BOK in WT and Nlrp6 -/- mouse glomeruli with or without HFrD, ( n = 6). (G) Western blot detection of BOK in MPC5, which was stimulated with or without 5 mM fructose after being transfected with siRNA-NC or siRNA- Nlrp6 , ( n = 6-8). (H) Western blot detection of BOK in PMPCs isolated from WT and Nlrp6 -/- mice, which were stimulated with or without 5 mM fructose, ( n = 6). (I) Representative IF images of TUNEL assay in WT and Nlrp6 -/- mouse glomeruli with or without HFrD, Scale: 20 μm. (J) Flow cytometry analysis of apoptotic cells through Annexin V-FITC/PI staining. MPC5 were stimulated with or without 5 mM fructose after being transfected with siRNA-NC or siRNA- Nlrp6 , ( n = 6). (K) Representative IF images of TUNEL assay in MPC5, which was stimulated with or without 5 mM fructose after being transfected with siRNA-NC or siRNA- Nlrp6 , Scale: 100 μm. (L) TEM images and quantification of podocyte apoptotic morphology and mitochondria in WT and Nlrp6 -/- mouse glomeruli with or without HFrD. Scale: 1 μm (Top), 250 nm (Bottom), ( n = 3). (M) Flow cytometry analysis of mitochondrial membrane potentials using JC-10 dye. MPC5 were stimulated with or without 5 mM fructose after being transfected with siRNA-NC or siRNA- Nlrp6 , ( n = 4). (N) OCR detection in MPC5, which was stimulated with or without 5 mM fructose after being transfected with siRNA-NC or siRNA- Nlrp6 , ( n = 4-6). (O)Western blot detection of cleaved Caspase 3 and cleaved Caspase 9 in WT and Nlrp6 -/- mouse glomeruli with or without HFrD, ( n = 6-8). (P) Western blot detection of cleaved Caspase 3 and cleaved Caspase 9 in MPC5, which were stimulated with or without 5 mM fructose after being transfected with siRNA-NC or siRNA- Nlrp6 , ( n = 6). (Q) Western blot detection of Cyto C performed on mitochondrial and cytosolic fractions in WT and Nlrp6 -/- mouse glomeruli with or without HFrD, ( n = 3). (R) Western blot detection of Cyto C was performed on mitochondrial and cytosolic fractions in MPC5, which were stimulated with or without 5 mM fructose after being transfected with siRNA-NC or siRNA- Nlrp6 , ( n = 3). (S) IF analysis of Cyto C and TOMM20 in MPC5, which were stimulated with or without 5 mM fructose after being transfected with siRNA-NC or siRNA- Nlrp6 . Scale: 25 μm. CASP: Caspase; PI: propidium iodide; TOMM20, translocase of the outer membrane 20; Cyto C: Cytochrome C. Data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Podocyte Caspase 3 activity level was assessed by the commercially available kit (C1168M, Beyotime).

Techniques: Inhibition, RNA Sequencing, Isolation, Western Blot, Transfection, TUNEL Assay, Flow Cytometry, Staining, Membrane

Journal: International Journal of Biological Sciences

Article Title: Gastrodin alleviates high fructose-induced podocyte mitochondria-mediated apoptosis by inhibiting NLRP6 to facilitate TRIM7-triggered Bok mRNA degradation

doi: 10.7150/ijbs.120307

Figure Lengend Snippet: NLRP6 inhibition relieves mitochondria-mediated apoptosis by downregulating BOK in high fructose-stimulated podocytes. (A) Western blot detection of BAK, BAX and Bcl-2 in WT and Nlrp6 -/ - mouse glomeruli with or without HFrD, ( n = 6-7). (B) Western blot detection of BAK, BAX and Bcl-2 in MPC5, which were stimulated with or without 5 mM fructose after being transfected with siRNA-NC or siRNA- Nlrp6 , ( n = 6). (C) Western blot detection of cleaved Caspase 3 and cleaved Caspase 9 in MPC5, which were stimulated with or without 5 mM fructose after being transfected with siRNA-NC or siRNA- Bok , ( n = 4-6). (D) Flow cytometry analysis of activated Caspase 3 in MPC5, which were stimulated with or without 5 mM fructose after being transfected with siRNA-NC or siRNA- Bok , ( n = 3). (E) OCR detection in MPC5, which was stimulated with or without 5 mM fructose after being transfected with siRNA-NC or siRNA- Bok , ( n = 6). (F) Flow cytometry analysis of total intracellular Ca 2+ concentration using Fluo-4 AM (1 μM) in MPC5, which were stimulated with or without 5 mM fructose after being transfected with siRNA-NC or siRNA- Bok , ( n = 4). (G) Western blot detection of Cyto C was performed on mitochondrial or cytosolic fractions in MPC5, which were stimulated with or without 5 mM fructose after being transfected with siRNA-NC or siRNA- Bok , ( n = 5). (H) Flow cytometry analysis of mitochondrial membrane potentials using JC-10 dye in MPC5, which were stimulated with or without 5 mM fructose after being transfected with siRNA-NC or siRNA- Bok , ( n = 3). (I) Representative IF images of TUNEL assay in MPC5, which were stimulated with or without 5 mM fructose after being transfected with siRNA-NC or siRNA- Bok , Scale: 100 μm. (J) Flow cytometry analysis of apoptotic cells through Annexin V-FITC/PI staining. MPC5 were stimulated with or without 5 mM fructose after being transfected with siRNA-NC or siRNA- Bok , ( n = 4). Data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. no significance, ns.

Article Snippet: Podocyte Caspase 3 activity level was assessed by the commercially available kit (C1168M, Beyotime).

Techniques: Inhibition, Western Blot, Transfection, Flow Cytometry, Concentration Assay, Membrane, TUNEL Assay, Staining

Journal: International Journal of Biological Sciences

Article Title: Gastrodin alleviates high fructose-induced podocyte mitochondria-mediated apoptosis by inhibiting NLRP6 to facilitate TRIM7-triggered Bok mRNA degradation

doi: 10.7150/ijbs.120307

Figure Lengend Snippet: NLRP6 downregulation reduces podocyte mitochondrial ROS and apoptosis via improving FAM213A antioxidant activity. (A) Western blot detection of FAM213A in WT and Nlrp6 -/- mouse glomeruli with or without HFrD, ( n = 6-8). (B) Western blot detection of FAM213A in MPC5, which was stimulated with or without 5 mM fructose after being transfected with siRNA-NC or siRNA- Nlrp6 , ( n = 5). (C) Western blot detection of FAM213A in PMPCs isolated from WT and Nlrp6 -/- mice, which were stimulated with or without 5 mM fructose, ( n = 4). (D) Western blot detection of FAM213A in MPC5, which were transfected with stimulated with or without 5 mM fructose after being transfected with siRNA-NC or siRNA- Bok , ( n = 4). (E) Western blot detection of cleaved Caspase 3 and cleaved Caspase 9 in MPC5 transfected with vector or Bok-HA or Fam213a-Flag , ( n = 5). (F) Flow cytometry analysis of activated Caspase 3 in MPC5 transfected with vector or Bok-HA or Fam213a-Flag , ( n = 5). (G) Western blot detection of Cyto C performed on mitochondrial and cytosolic fractions in MPC5 transfected with vector or Bok-HA or Fam213a-Flag , ( n = 3). (H) OCR detection in MPC5 transfected with vector or Bok-HA or Fam213a-Flag , ( n = 4). (I) ROS levels were considered as the fluorescence intensity of fluorogenic probe DCFH 2 -DA, measured via microplate reader in MPC5 transfected with vector or Bok-HA or Fam213a-Flag , ( n = 18). (J) Mitochondrial ROS production was considered as the fluorescence intensity of labeling fluorogenic probe MitoSOX, measured by flow cytometry in MPC5 transfected with vector or Bok-HA or Fam213a-Flag , ( n = 6). (K) Flow cytometry analysis of mitochondrial membrane potentials using JC-10 dye in MPC5 transfected with vector or Bok-HA or Fam213a-Flag , ( n = 4). (L) Representative IF images of TUNEL assay in MPC5 transfected with vector or Bok-HA or Fam213a-Flag , Scale: 100 μm. (M) Flow cytometry analysis of apoptotic cells through Annexin V-FITC/PI staining in MPC5 transfected with vector or Bok-HA or Fam213a-Flag , ( n = 5). Data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Podocyte Caspase 3 activity level was assessed by the commercially available kit (C1168M, Beyotime).

Techniques: Antioxidant Activity Assay, Western Blot, Transfection, Isolation, Plasmid Preparation, Flow Cytometry, Fluorescence, Labeling, Membrane, TUNEL Assay, Staining

Journal: International Journal of Biological Sciences

Article Title: Gastrodin alleviates high fructose-induced podocyte mitochondria-mediated apoptosis by inhibiting NLRP6 to facilitate TRIM7-triggered Bok mRNA degradation

doi: 10.7150/ijbs.120307

Figure Lengend Snippet: Pharmacological inhibition of NLRP6 by gastrodin ameliorates high fructose-induced glomerular podocyte mitochondria-mediated apoptosis. (A) Molecular docking shows interactions between gastrodin and NLRP6. The hydrogen-bonding interactions are shown in yellow dashed lines. (B) The binding affinity of gastrodin to NLRP6 was measured by MST, ( n = 3). (C) Western blot detection of NLRP6 in control mouse and HFrD-fed mouse glomeruli with or without gastrodin treatment (25, 50, and 100 mg/kg), ( n = 6). (D) Western blot detection of NLRP6 in MPC5, which was stimulated with 5 mM fructose as well as gastrodin (25, 50, and 100 μM) or not, ( n = 5-6). (E) Western blot detection of TRIM7, BOK, and FAM213A in control mouse and HFrD-fed mouse glomeruli with or without gastrodin treatment (25, 50, and 100 mg/kg), ( n = 6). (F) Western blot detection of TRIM7, BOK, and FAM213A in MPC5, which were stimulated with 5 mM fructose as well as gastrodin (25, 50, and 100 μM) or not, ( n = 6-8). (G) Western blot detection of cleaved Caspase 3 and cleaved Caspase 9 in control mouse and HFrD-fed mouse glomeruli with or without gastrodin treatment (25, 50, and 100 mg/kg), ( n = 6-7). (H) Western blot detection of cleaved Caspase 3 and cleaved Caspase 9 in MPC5, which were stimulated with 5 mM fructose as well as gastrodin (25, 50, and 100 μM) or not, ( n = 5). (I) Flow cytometry analysis of activated Caspase 3 in MPC5, which were stimulated with 5 mM fructose as well as gastrodin (25, 50, and 100 μM) or not, ( n = 4). (J) ROS levels were considered as the fluorescence intensity of fluorogenic probe DCFH 2 -DA, measured via microplate reader in MPC5. MPC5 were stimulated with 5 mM fructose as well as gastrodin (25, 50, and 100 μM) or not, ( n = 10). (K) Mitochondrial ROS production was considered as the fluorescence intensity of fluorogenic probe MitoSOX, measured by flow cytometry in MPC5. MPC5 were stimulated with 5 mM fructose as well as gastrodin (25, 50, and 100 μM) or not, ( n = 3-4). (L) OCR detection in MPC5, which were stimulated with 5 mM fructose as well as gastrodin (25, 50, and 100 μM) or not, ( n = 8). (M) TEM images and quantification of podocyte in control mouse and HFrD-fed mouse glomeruli with or without gastrodin treatment (25, 50, and 100 mg/kg). Scale: 1 μm (Top), 250 nm (Bottom), ( n = 3). (N) Western blot detection of Cyto C performed on mitochondrial and cytosolic fractions in control mouse and HFrD-fed mouse glomeruli with or without gastrodin treatment (25, 50, and 100 mg/kg), ( n = 3). (O) Western blot detection of Cyto C was performed on mitochondrial and cytosolic fractions in MPC5, which were stimulated with 5 mM fructose as well as gastrodin (25, 50, and 100 μM) or not, ( n = 3). (P) Flow cytometry analysis of mitochondrial membrane potentials using JC-10 dye in MPC5, which were stimulated with 5 mM fructose as well as gastrodin (25, 50, and 100 μM) or not, ( n = 4). (Q) Representative IF images of TUNEL assay in control mouse and HFrD-fed mouse glomeruli with or without the treatment of gastrodin (25, 50, and 100 mg/kg), Scale: 20 μm. (R) Flow cytometry analysis of apoptotic cells through Annexin V-FITC/PI staining in MPC5, which were stimulated with 5 mM fructose as well as gastrodin (25, 50, and 100 μM) or not, ( n = 5). (S) Representative IF images of TUNEL assay in MPC5, which were stimulated with 5 mM fructose as well as gastrodin (25, 50, and 100 μM) or not, Scale: 100 μm. Data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Podocyte Caspase 3 activity level was assessed by the commercially available kit (C1168M, Beyotime).

Techniques: Inhibition, Binding Assay, Western Blot, Control, Flow Cytometry, Fluorescence, Membrane, TUNEL Assay, Staining