Journal: British Journal of Pharmacology

Article Title: Connexin32 ameliorates renal fibrosis in diabetic mice by promoting K48‐linked NADPH oxidase 4 polyubiquitination and degradation

doi: 10.1111/bph.14853

Figure Lengend Snippet: Overexpression of Cx32 ameliorates renal function and fibrosis in STZ‐induced diabetic mice. (a) Glomerular histopathology analysis was performed by periodic acid‐Schiff (PAS) staining (400× magnification). (b,c) The FBG and body weight of diabetic mice were analysed. (d) The expression levels of fibronectin (FN) and Cx32 in the glomeruli were shown by immunohistochemical staining (400× magnification). (e) The kidney weight/body weight ratio was calculated to evaluate the kidney hypertrophy index. (f) Serum creatinine (Cr) was detected to assess renal injury. (g) The expression of Cx32 and fibronectin in the kidney of mice were measured using the western blot assay. (h,i) Serum BUN and 24‐hr UP were detected to assess renal injury. The data are presented as the means ± SD; n = 7 (two outliers were excluded). # P < .05, significantly different from Ctrl + Ad‐V; * P < .05, significantly different from STZ + Ad‐V. Ad‐Cx32, Cx32 adenovirus; Ad‐V, vector adenovirus; BUN, blood urea nitrogen; Cr, serum creatinine; Ctrl, control; FBG, fasting blood glucose; STZ, streptozocin; UP 24 hr, urine protein for 24 hr

Article Snippet: An enhanced chemiluminescence substrate (Cat# WP20005) for the detection of HRP was obtained from Thermo Fisher Scientific, Inc. Antibodies against fibronectin (FN; Proteintech Cat# 15613‐1‐AP, RRID:AB_2105691), Nox4 (Proteintech Cat# 14347‐1‐AP, RRID:AB_10638146), α smooth muscle actin (α‐SMA; Proteintech Cat# 14395‐1‐AP, RRID:AB_2223009), TGF‐β1 (Proteintech Cat# 21898‐1‐AP, RRID:AB_2811115), and Smurf1 (Proteintech Cat# 55175‐1‐AP, RRID:AB_10859079) were purchased from Proteintech Group (Chicago, IL, USA); α‐Tubulin (Sigma‐Aldrich Cat# T8203, RRID:AB_1841230) was purchased from Sigma–Aldrich Corporation; Cx32 (Abcam Cat# ab66613, RRID:AB_1141052) was from Abcam (Cambridge, MA, USA); Cx32 (Novus Cat# NBP2‐53381, RRID:AB_2811114) was from Novus Biologicals (Centennial, USA); and ubiquitin (Ub; Cell Signaling Technology Cat# 3936, RRID:AB_331292) was from Cell Signaling Technology (Danvers, USA).

Techniques: Over Expression, Histopathology, Staining, Expressing, Immunohistochemical staining, Western Blot, Plasmid Preparation

Journal: British Journal of Pharmacology

Article Title: Connexin32 ameliorates renal fibrosis in diabetic mice by promoting K48‐linked NADPH oxidase 4 polyubiquitination and degradation

doi: 10.1111/bph.14853

Figure Lengend Snippet: Cx32 deficiency exacerbates renal fibrosis in STZ‐induced diabetic mice. (a) Glomerular histopathology analysis by periodic acid‐Schiff (PAS) staining (400× magnification). (b,c) The FBG and body weight of diabetic mice were analysed. (d) The expression of fibronectin (FN) and Cx32 in the glomeruli were shown by immunohistochemical staining (400× magnification). (e) The kidney weight/body weight ratio was calculated to evaluate the kidney hypertrophy index. (f) Serum Cr was detected to assess renal injury. (g) The expression of fibronectin in the kidney of mice were measured by western blot assay. (h,i) Serum BUN and 24‐hr UP were assayed to assess renal injury. The data are presented as the means ± SD; n = 6 (two outliers were excluded). # P < .05, significantly different from WT; * P < .05, significantly different from WT + STZ. BUN, blood urea nitrogen; Cr, serum creatinine; FBG, fasting blood glucose; KO, knockout; STZ, streptozocin; UP 24 hr, urine protein for 24 hr; WT, wild type

Article Snippet: An enhanced chemiluminescence substrate (Cat# WP20005) for the detection of HRP was obtained from Thermo Fisher Scientific, Inc. Antibodies against fibronectin (FN; Proteintech Cat# 15613‐1‐AP, RRID:AB_2105691), Nox4 (Proteintech Cat# 14347‐1‐AP, RRID:AB_10638146), α smooth muscle actin (α‐SMA; Proteintech Cat# 14395‐1‐AP, RRID:AB_2223009), TGF‐β1 (Proteintech Cat# 21898‐1‐AP, RRID:AB_2811115), and Smurf1 (Proteintech Cat# 55175‐1‐AP, RRID:AB_10859079) were purchased from Proteintech Group (Chicago, IL, USA); α‐Tubulin (Sigma‐Aldrich Cat# T8203, RRID:AB_1841230) was purchased from Sigma–Aldrich Corporation; Cx32 (Abcam Cat# ab66613, RRID:AB_1141052) was from Abcam (Cambridge, MA, USA); Cx32 (Novus Cat# NBP2‐53381, RRID:AB_2811114) was from Novus Biologicals (Centennial, USA); and ubiquitin (Ub; Cell Signaling Technology Cat# 3936, RRID:AB_331292) was from Cell Signaling Technology (Danvers, USA).

Techniques: Histopathology, Staining, Expressing, Immunohistochemical staining, Western Blot, Knock-Out

Journal: British Journal of Pharmacology

Article Title: Connexin32 ameliorates renal fibrosis in diabetic mice by promoting K48‐linked NADPH oxidase 4 polyubiquitination and degradation

doi: 10.1111/bph.14853

Figure Lengend Snippet: Regulation of fibronectin expression by Cx32 in GMCs is not dependent on GJIC. (a) The expression of Cx32 and GFP‐tag were measured by western blotting to confirm that the plasmids were successfully transfected into GMCs. (b) Cx32 overexpression inhibited fibronectin expression in HG‐induced GMCs. The transfection of Cx32 siRNA decreased Cx32 (c) expression and up‐regulated fibronectin (d) expression. (e) Photomicrographs obtained after Lucifer yellow scrape loading in GMCs transfected with the Cx32 plasmid with or without 18‐α‐GA (10 μM). (f) Treatment with 18‐α‐GA did not block the down‐regulation of fibronectin expression by Cx32. The data are presented as the means ± SD; n = 5. # P < .05, significantly different from NG; * P < .05, significantly different from HG,

Article Snippet: An enhanced chemiluminescence substrate (Cat# WP20005) for the detection of HRP was obtained from Thermo Fisher Scientific, Inc. Antibodies against fibronectin (FN; Proteintech Cat# 15613‐1‐AP, RRID:AB_2105691), Nox4 (Proteintech Cat# 14347‐1‐AP, RRID:AB_10638146), α smooth muscle actin (α‐SMA; Proteintech Cat# 14395‐1‐AP, RRID:AB_2223009), TGF‐β1 (Proteintech Cat# 21898‐1‐AP, RRID:AB_2811115), and Smurf1 (Proteintech Cat# 55175‐1‐AP, RRID:AB_10859079) were purchased from Proteintech Group (Chicago, IL, USA); α‐Tubulin (Sigma‐Aldrich Cat# T8203, RRID:AB_1841230) was purchased from Sigma–Aldrich Corporation; Cx32 (Abcam Cat# ab66613, RRID:AB_1141052) was from Abcam (Cambridge, MA, USA); Cx32 (Novus Cat# NBP2‐53381, RRID:AB_2811114) was from Novus Biologicals (Centennial, USA); and ubiquitin (Ub; Cell Signaling Technology Cat# 3936, RRID:AB_331292) was from Cell Signaling Technology (Danvers, USA).

Techniques: Expressing, Western Blot, Transfection, Over Expression, Plasmid Preparation, Blocking Assay

Journal: British Journal of Pharmacology

Article Title: Connexin32 ameliorates renal fibrosis in diabetic mice by promoting K48‐linked NADPH oxidase 4 polyubiquitination and degradation

doi: 10.1111/bph.14853

Figure Lengend Snippet: Cx32 decreases oxidative stress in the kidney of diabetic mice and HG‐induced GMCs. Treatment with Cx32 adenovirus increased total SOD activity (a) and decreased the MDA levels (b) in the kidneys of diabetic mice. The data are presented as the means ± SD; n = 7. # P < .05, significantly different from Ctrl + Ad‐V; * P < .05, significantly different from STZ + Ad‐V. Cx32 deficiency decreased total SOD activity (c) and increased the MDA levels (d) in the kidney of diabetic mice. The data are presented as the means ± SD; n = 6. # P < .05, significantly different from WT; * P < .05, significantly different from WT + STZ. KO, knockout; WT, wild type. (e) The hydrogen peroxide released from HG‐cultured GMCs was measured using Amplex Red. Overexpression of Cx32 inhibited the generation of hydrogen peroxide in GMCs induced by HG for 12 hr. (f) The mitochondrial superoxide levels were detected using the MitoSOX Red probe. The data are presented as the means ± SD; n = 5. # P < .05, significantly different from NG; * P < .05, significantly different from HG. Ad‐Cx32, Cx32 adenovirus; Ad‐V, vector adenovirus; Ctrl, control; MDA, malonaldehyde; STZ, streptozocin.

Article Snippet: An enhanced chemiluminescence substrate (Cat# WP20005) for the detection of HRP was obtained from Thermo Fisher Scientific, Inc. Antibodies against fibronectin (FN; Proteintech Cat# 15613‐1‐AP, RRID:AB_2105691), Nox4 (Proteintech Cat# 14347‐1‐AP, RRID:AB_10638146), α smooth muscle actin (α‐SMA; Proteintech Cat# 14395‐1‐AP, RRID:AB_2223009), TGF‐β1 (Proteintech Cat# 21898‐1‐AP, RRID:AB_2811115), and Smurf1 (Proteintech Cat# 55175‐1‐AP, RRID:AB_10859079) were purchased from Proteintech Group (Chicago, IL, USA); α‐Tubulin (Sigma‐Aldrich Cat# T8203, RRID:AB_1841230) was purchased from Sigma–Aldrich Corporation; Cx32 (Abcam Cat# ab66613, RRID:AB_1141052) was from Abcam (Cambridge, MA, USA); Cx32 (Novus Cat# NBP2‐53381, RRID:AB_2811114) was from Novus Biologicals (Centennial, USA); and ubiquitin (Ub; Cell Signaling Technology Cat# 3936, RRID:AB_331292) was from Cell Signaling Technology (Danvers, USA).

Techniques: Activity Assay, Knock-Out, Cell Culture, Over Expression, Plasmid Preparation

Journal: British Journal of Pharmacology

Article Title: Connexin32 ameliorates renal fibrosis in diabetic mice by promoting K48‐linked NADPH oxidase 4 polyubiquitination and degradation

doi: 10.1111/bph.14853

Figure Lengend Snippet: Nox4, at least partly, mediates the down‐regulation of fibronectin expression by Cx32 in HG‐induced GMCs. (a and b) The expression of Nox4 in the glomeruli was shown by immumohistochemical staining (400× magnification). (c) Cx32 overexpression decreased the expression of Nox4. # P < .05, significantly different from NG; * P < .05, significantly different from HG. (d) Cx32 knockdown increased Nox4 expression. # P < .05, significantly different from NG; * P < .05, significantly different from HG. (e) Cx32 co‐localized with Nox4 in GMCs under normal conditions, as revealed by immunofluorescence assay. Red fluorescence indicates the localization of Cx32. Green fluorescence indicates Nox4. Blue fluorescence indicates nuclei. (f) Immunoprecipitation analysis was used to detect the interaction between Cx32 and Nox4 in GMCs. (g) Overexpression of Nox4 under HG conditions inhibited the down‐regulation effects of Cx32 in the expression of fibronectin . # P < .05, significantly different from NG; * P < .05, significantly different from HG; $ P < .05, significantly different from HG + Cx32. The data are presented as the means ± SD; n = 5.

Article Snippet: An enhanced chemiluminescence substrate (Cat# WP20005) for the detection of HRP was obtained from Thermo Fisher Scientific, Inc. Antibodies against fibronectin (FN; Proteintech Cat# 15613‐1‐AP, RRID:AB_2105691), Nox4 (Proteintech Cat# 14347‐1‐AP, RRID:AB_10638146), α smooth muscle actin (α‐SMA; Proteintech Cat# 14395‐1‐AP, RRID:AB_2223009), TGF‐β1 (Proteintech Cat# 21898‐1‐AP, RRID:AB_2811115), and Smurf1 (Proteintech Cat# 55175‐1‐AP, RRID:AB_10859079) were purchased from Proteintech Group (Chicago, IL, USA); α‐Tubulin (Sigma‐Aldrich Cat# T8203, RRID:AB_1841230) was purchased from Sigma–Aldrich Corporation; Cx32 (Abcam Cat# ab66613, RRID:AB_1141052) was from Abcam (Cambridge, MA, USA); Cx32 (Novus Cat# NBP2‐53381, RRID:AB_2811114) was from Novus Biologicals (Centennial, USA); and ubiquitin (Ub; Cell Signaling Technology Cat# 3936, RRID:AB_331292) was from Cell Signaling Technology (Danvers, USA).

Techniques: Expressing, Staining, Over Expression, Immunofluorescence, Fluorescence, Immunoprecipitation

Journal: British Journal of Pharmacology

Article Title: Connexin32 ameliorates renal fibrosis in diabetic mice by promoting K48‐linked NADPH oxidase 4 polyubiquitination and degradation

doi: 10.1111/bph.14853

Figure Lengend Snippet: Cx32 decreases the protein levels of Nox4 by promoting K48‐linked polyubiquitination. (a) Co‐transfection of the plasmids pcDNA3.1‐Nox4 and HA‐Ub (wild type) or HA‐Ub‐K0 (mutant type) in HEK‐293A cells and western blotting results showed that Nox4 could be polyubiquitinated. (b) The plasmid pcDNA3.1‐Nox4 was co‐transfected with plasmids HA‐Ub or K48‐only ubiquitin (HA‐Ub‐K48) or K63‐only ubiquitin (HA‐Ub‐K63) in HEK‐293A cells exposed to MG132 (5 μM). (c) Overexpression of Cx32 increased the polyubiquitination of Nox4 in GMCs induced by HG.

Article Snippet: An enhanced chemiluminescence substrate (Cat# WP20005) for the detection of HRP was obtained from Thermo Fisher Scientific, Inc. Antibodies against fibronectin (FN; Proteintech Cat# 15613‐1‐AP, RRID:AB_2105691), Nox4 (Proteintech Cat# 14347‐1‐AP, RRID:AB_10638146), α smooth muscle actin (α‐SMA; Proteintech Cat# 14395‐1‐AP, RRID:AB_2223009), TGF‐β1 (Proteintech Cat# 21898‐1‐AP, RRID:AB_2811115), and Smurf1 (Proteintech Cat# 55175‐1‐AP, RRID:AB_10859079) were purchased from Proteintech Group (Chicago, IL, USA); α‐Tubulin (Sigma‐Aldrich Cat# T8203, RRID:AB_1841230) was purchased from Sigma–Aldrich Corporation; Cx32 (Abcam Cat# ab66613, RRID:AB_1141052) was from Abcam (Cambridge, MA, USA); Cx32 (Novus Cat# NBP2‐53381, RRID:AB_2811114) was from Novus Biologicals (Centennial, USA); and ubiquitin (Ub; Cell Signaling Technology Cat# 3936, RRID:AB_331292) was from Cell Signaling Technology (Danvers, USA).

Techniques: Cotransfection, Mutagenesis, Western Blot, Plasmid Preparation, Transfection, Over Expression

Journal: British Journal of Pharmacology

Article Title: Connexin32 ameliorates renal fibrosis in diabetic mice by promoting K48‐linked NADPH oxidase 4 polyubiquitination and degradation

doi: 10.1111/bph.14853

Figure Lengend Snippet: Diagram of the pathways involved in the effects of Cx32 on kidneys of diabetic mice. Cx32 ameliorates diabetic renal fibrosis by promoting Nox4 polyubiquitination and degradation via the inhibition of Smurf1 expression. Under normal conditions, Smurf1 is bound to the carboxyl terminal of Cx32 on the membrane of GMCs. Also, Nox4 is mainly modified by K48‐linked polyubiquitination and subsequently degraded by the proteasome. Under diabetic or HG conditions, the protein expression of Cx32 was decreased, leading to the up‐regulation of Smurf1 expression. Overexpression of Smurf1 reduces K48‐linked polyubiquitination of Nox4 and increases its protein levels, resulting in intracellular H2O2 overproduction. Increased oxidative stress further promoted extracellular matrix protein (fibronectin) expression, leading to exacerbation of diabetic renal fibrosis

Article Snippet: An enhanced chemiluminescence substrate (Cat# WP20005) for the detection of HRP was obtained from Thermo Fisher Scientific, Inc. Antibodies against fibronectin (FN; Proteintech Cat# 15613‐1‐AP, RRID:AB_2105691), Nox4 (Proteintech Cat# 14347‐1‐AP, RRID:AB_10638146), α smooth muscle actin (α‐SMA; Proteintech Cat# 14395‐1‐AP, RRID:AB_2223009), TGF‐β1 (Proteintech Cat# 21898‐1‐AP, RRID:AB_2811115), and Smurf1 (Proteintech Cat# 55175‐1‐AP, RRID:AB_10859079) were purchased from Proteintech Group (Chicago, IL, USA); α‐Tubulin (Sigma‐Aldrich Cat# T8203, RRID:AB_1841230) was purchased from Sigma–Aldrich Corporation; Cx32 (Abcam Cat# ab66613, RRID:AB_1141052) was from Abcam (Cambridge, MA, USA); Cx32 (Novus Cat# NBP2‐53381, RRID:AB_2811114) was from Novus Biologicals (Centennial, USA); and ubiquitin (Ub; Cell Signaling Technology Cat# 3936, RRID:AB_331292) was from Cell Signaling Technology (Danvers, USA).

Techniques: Inhibition, Expressing, Membrane, Modification, Over Expression

Journal: British Journal of Pharmacology

Article Title: Connexin32 ameliorates renal fibrosis in diabetic mice by promoting K48‐linked NADPH oxidase 4 polyubiquitination and degradation

doi: 10.1111/bph.14853

Figure Lengend Snippet: Cx32 increases K48‐linked polyubiquitination of Nox4 via the inhibition of Smurf1 expression. (a) Cx32 overexpression decreased the expression of Smurf1, # P < .05, significantly different from NG; * P < .05, significantly different from HG. (b) Cx32 knockdown increased Smurf1 expression, # P < .05, significantly different from NG; * P < .05, significantly different from HG. (c) Cx32 interacted with Smurf1 in GMCs. (d) Smurf1 increased Nox4 expression through its HECT domain, # P < .05, significantly different from NG; * P < .05, significantly different from Smurf1. The data are presented as the means ± SD; n = 5. (e) Co‐transfection of plasmids HA‐Ub‐K48 and pRK‐Myc‐Smurf1 was performed in GMCs

Article Snippet: An enhanced chemiluminescence substrate (Cat# WP20005) for the detection of HRP was obtained from Thermo Fisher Scientific, Inc. Antibodies against fibronectin (FN; Proteintech Cat# 15613‐1‐AP, RRID:AB_2105691), Nox4 (Proteintech Cat# 14347‐1‐AP, RRID:AB_10638146), α smooth muscle actin (α‐SMA; Proteintech Cat# 14395‐1‐AP, RRID:AB_2223009), TGF‐β1 (Proteintech Cat# 21898‐1‐AP, RRID:AB_2811115), and Smurf1 (Proteintech Cat# 55175‐1‐AP, RRID:AB_10859079) were purchased from Proteintech Group (Chicago, IL, USA); α‐Tubulin (Sigma‐Aldrich Cat# T8203, RRID:AB_1841230) was purchased from Sigma–Aldrich Corporation; Cx32 (Abcam Cat# ab66613, RRID:AB_1141052) was from Abcam (Cambridge, MA, USA); Cx32 (Novus Cat# NBP2‐53381, RRID:AB_2811114) was from Novus Biologicals (Centennial, USA); and ubiquitin (Ub; Cell Signaling Technology Cat# 3936, RRID:AB_331292) was from Cell Signaling Technology (Danvers, USA).

Techniques: Inhibition, Expressing, Over Expression, Cotransfection