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 expression is increased in high fructose-induced glomerular podocyte injury in mice (A) Nlrp 6 and Nlrp12 mRNA levels in WT mouse glomeruli, ( n = 6). (B) Nlrp 6 and Nlrp12 mRNA levels in WT mouse glomeruli with or without HFrD, ( n = 4-6). (C) The representative micrographs of RNAscope ( Nlrp6 mRNA, cyan) and IF (Podocin, green; Synaptopodin, red) co-labeled the podocytes of mouse glomeruli. Scale: 25 μm. (D) Western blot detection of NLRP6 in WT mouse glomeruli with or without HFrD, (n = 7). (E) Representative IF images of NLRP6 (green) and Synaptopodin (red) co-labeled the podocytes of mouse glomeruli. Scale: 30 μm. (F) Western blot detection of NLRP6 in kidney cells, ( n = 4). (G) Western blot detection of NLRP6 in kidney cells exposed to 5 mM fructose, ( n = 4). (H) Western blot detection of NLRP6 in podocytes (HPCs, MPC5, PMPCs) exposed to 5 mM fructose or not, ( n = 4-6). (I) Representative IF images of NLRP6 (green) in podocytes (HPCs, MPC5, PMPC) exposed to 5 mM fructose or not. HPCs, Scale: 20 μm; MPC5, Scale: 20 μm; PMPC, Scale: 20 μm. NC: normal chow; HFrD: high fructose diet; HPCs: human podocytes; MPC5: mouse podocyte clone-5 cell; HGMC: human glomerular mesangial cell; PMGEC: primary mouse glomerular endothelial cell; HK-2: human proximal tubule epithelial cell; PMPC: primary mouse podocyte. Data are expressed as the mean ± SEM. ** P < 0.01, *** P < 0.001.

Article Snippet: The intracellular Ca 2+ concentration of podocytes were measured by the Fluo-4 AM probe (HY-101896, MedChemExpress).

Techniques: Expressing, RNAscope, Labeling, Western Blot

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: Knockout of Nlrp6 alleviates high fructose-induced glomerular podocyte injury in mice. (A) A schematic diagram shows the construction of Nlrp6 -/- mouse (top) and a diagram of the procedure of HFrD-fed mouse (bottom). (B-C) The kidney wet weight, and kidney index of WT and Nlrp6 -/- mouse with or without HFrD, ( n = 7). (D-G) Serum uric acid, creatinine, urea nitrogen, and the ratio of urine albumin to creatinine were measured in WT and Nlrp6 -/- mice with or without HFrD, ( n = 4-15), respectively. (H-L) Representative images show glomerular changes by morphological examinations, including PAS staining, HE staining, Masson staining, and TEM in WT and Nlrp6 -/- mice with or without HFrD. Scale: PAS, HE, Masson: 20 μm; TEM: 1 μm. (M) Representative images of IF and quantifications of Synaptopodin (red), Podocin(green), and Nephrin (magenta) in WT and Nlrp6 -/- mouse glomeruli with or without HFrD. Scale: 25 μm. (N) Western blot detection of Podocin and Nephrin in WT and Nlrp6 -/- mouse glomeruli with or without HFrD, ( n = 7-8). (O) Western blot detection of Podocin and Nephrin in PMPCs isolated from WT and Nlrp6 -/- mice, which were stimulated with 5 mM fructose or not, ( n = 4). (P) Western blot detection of Podocin and Nephrin in MPC5, which were transfected with siRNA-NC or siRNA- Nlrp6 , subsequently stimulated with 5 mM fructose or not, ( n = 6). WT: Wild type; Nlrp6 -/- : Nlr p6 knockout; KW/BW: kidney weight/body weight; GBM: glomerular basement membrane; Ctrl: Control; Fru: Fructose; si-NC: siRNA-Negative control; si- Nlrp 6: siRNA- Nlrp 6. Data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The intracellular Ca 2+ concentration of podocytes were measured by the Fluo-4 AM probe (HY-101896, MedChemExpress).

Techniques: Knock-Out, Staining, Western Blot, Isolation, Transfection, Membrane, Control, Negative Control

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: The intracellular Ca 2+ concentration of podocytes were measured by the Fluo-4 AM probe (HY-101896, MedChemExpress).

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: The intracellular Ca 2+ concentration of podocytes were measured by the Fluo-4 AM probe (HY-101896, MedChemExpress).

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 inhibition upregulates TRIM7 to decrease Bok mRNA stability in podocytes. (A) Western blot detection of TRIM7 expression in WT and Nlrp6 -/- mouse glomeruli with or without HFrD, ( n =6). (B) Western blot detection of TRIM7 in MPC5, which was stimulated with or without 5 mM fructose after being transfected with siRNA-NC or siRNA- Nlrp6 , ( n = 6). (C) Western blot detection of TRIM7 in PMPCs isolated from WT and Nlrp6 -/- mice, which were stimulated with or without 5 mM fructose after being transfected with siRNA-NC or siRNA- Nlrp6 , ( n = 6). (D) Bok mRNA level in MPC5, which was stimulated with or without 5 mM fructose after being transfected with vector or Trim7-Flag , ( n = 6). (E) Western blot detection of BOK in MPC5, which were stimulated with or without 5 mM fructose after being transfected with vector or Trim7-Flag , ( n = 4-5). (F-G) Western blot detection of BOK in MPC5 exposed to 5 μg/mL CHX or not for different times after being transfected with vector or Trim7-Flag , ( n = 3). (H) Bok remaining mRNA level in MPC5 exposed to 5 μg/mL Act D or not for different times after being transfected with vector or Trim7-Flag , ( n = 6-9). (I) The schematic diagram shows mouse Bok mRNA with AU-rich element (ARE). (J) RIP to assess the connection between Bok mRNA and TRIM7. Cell lysates from MPC5 transfected with vector or Trim7-Flag were immunoprecipitated with IgG or Flag-tag, The mRNA level of Bok recruited to TRIM7 was examined by qRT-PCR, ( n = 3-4). (K) RNA pull-down subsequently followed by Western blot. Biotin-labeled 3'UTR, CDS, and 5'UTR of Bok mRNA were incubated with MPC5 cell lysates. Following pull-down, the recruitment of TRIM7 to Bok mRNA was examined by Western blot. (L) Left, the schematic diagram shows the mutant sites of mouse Bok mRNA ARE region. Right, RNA pull-down was subsequently followed by Western blot. Biotin-labeled 3'UTR, 3'UTR mutant of Bok mRNA or Negative control transcript were incubated with MPC5 cell lysates. Following pull-down, the recruitment of TRIM7 to Bok mRNA was examined by Western blot. (M) Left, the schematic diagram shows different truncated mutants of mouse Trim7-Flag plasmid . Right, RIP to assess the connection between Bok mRNA and TRIM7 truncated mutants. Cell lysates from MPC5 transfected with Trim7-Flag truncated mutants were immunoprecipitated with IgG or Flag-tag. The mRNA level of Bok recruited to Trim7 truncated mutants was examined by qRT-PCR, ( n = 3-4). (N) Remaining Bok mRNA level in MPC5, which were transfected with vector or Trim7-Flag Full length or Trim7-Flag truncated mutants, and subsequently cultured with 5 μg/mL Act D after 12 h, ( n = 4). Vec: Vector; FL: Full length; T1: Truncated mutant 1; T2: Truncated mutant 2; T3: Truncated mutant 3; T4: Truncated mutant 4. Data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. no significance, ns.

Article Snippet: The intracellular Ca 2+ concentration of podocytes were measured by the Fluo-4 AM probe (HY-101896, MedChemExpress).

Techniques: Inhibition, Western Blot, Expressing, Transfection, Isolation, Plasmid Preparation, Immunoprecipitation, FLAG-tag, Quantitative RT-PCR, Labeling, Incubation, Mutagenesis, Negative Control, Cell Culture

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: The intracellular Ca 2+ concentration of podocytes were measured by the Fluo-4 AM probe (HY-101896, MedChemExpress).

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: The intracellular Ca 2+ concentration of podocytes were measured by the Fluo-4 AM probe (HY-101896, MedChemExpress).

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

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Modulation of the APOM/S1PR4 Pathway Reduces Podocyte Lipid Overload in Alport Syndrome via Distinct Autophagy and Efflux Mechanisms

doi: 10.1681/ASN.0000000996

Figure Lengend Snippet: (a) Representative Western blot and (b) bar graph quantification of APOM in scrambled control (shSC) and APOM knockdown (shAPOM) human podocytes (n=3). Bar graph quantification of (c) apoptosis and (d) cytotoxic cell death in shAPOM podocytes (n=4–5). (e) Confocal images of lipid droplets and (f) bar graph quantification of lipid droplet number in shAPOM podocytes (n = 3). HCS CellMask (green), DAPI (blue), HCS neutral LipidTox (red); 20× magnification. (g) Representative Western blot and (h) bar graph quantification of S1PR4 overexpressing (S1PR4 OE) (n=3). Bar graph quantification of (i) apoptosis and (j) cytotoxic cell death in S1PR4 OE podocytes (n=4). (k) Confocal images of lipid droplets and (l) bar graph quantification of lipid droplet number in S1PR4 OE podocytes (n = 3). Nile Red (green), DAPI (blue), CellMask HCS and phalloidin (red); 20× magnification.

Article Snippet: The knockdown control podocyte cell line was generated by infection with scrambled shRNA (Origene, TR30021V).

Techniques: Knockdown, Over Expression, Western Blot, Control