Journal: Cell Death & Disease

Article Title: β -Arrestins promote podocyte injury by inhibition of autophagy in diabetic nephropathy

doi: 10.1038/cddis.2016.89

Figure Lengend Snippet: Upregulation of β -arrestin-1 and β -arrestin-2 in the kidney from STZ-induced diabetic mice, diabetic db/db mice and kidney biopsies from diabetic patients. ( a ) Relative mRNA levels of Arrb1 and Arrb2 in the kidney from STZ-induced diabetic mice (mean±S.E.M.). ( b ) Representative western blotting gel documents and summarized data showing the relative protein levels of β -arrestin-1 and β -arrestin-2 in the kidney from STZ-induced diabetic mice (mean±S.E.M.). ( c ) Representative images are from ISH of β -arrestin-1 and β -arrestin-2 in the kidney from STZ-induced diabetic mice (arrowheads: representative podocytes). Scramble probes were used as negative controls. ( d ) Representative western blotting gel documents and summarized data showing the relative protein levels of β -arrestin-1/2 in the kidney from diabetic db/db mice and their genetic control db/+ mice (mean±S.E.M.). ( e ) Relative mRNA levels of Arrb1 in from normal subjects ( n =9) and patients with DN ( n =8) or DM-NN ( n =8) (mean±S.E.M.). ( f ) Negative correlation (Spearman r =−0.7471, P <0.05) between Arrb1 mRNA levels and estimated glomerular filtration rate (eGFR) in all subjects. ( g ) Relative mRNA levels of Arrb2 in the renal biopsies from different patients (mean±S.E.M.). ( h ) Negative correlation (Spearman r =−0.7845, P <0.05) between Arrb2 mRNA levels and eGFR in all subjects. * P <0.05 versus normal subjects

Article Snippet: Formalin-fixed, paraffin-embedded kidney tissues were sectioned (6- μ m thickness) and assayed for Arrb1 and Arrb2 RNA expression by ISH as described previously., , Arrb2 was detected by the Arrb2 mRNA ISH Kit with digoxigenin (DIG)-labeled probe (Boster Biological Technology Co., Wuhan, China) according to the manufacturer's protocols.

Techniques: Western Blot, Control, Filtration

Journal: Cell Death & Disease

Article Title: β -Arrestins promote podocyte injury by inhibition of autophagy in diabetic nephropathy

doi: 10.1038/cddis.2016.89

Figure Lengend Snippet: Either Arrb1 or Arrb2 deficiency ameliorated renal injury in diabetic mice. ( a ) Urinary ACR in different groups of mice. ( b ) Representative photomicrographs showing typical glomerular structure changes in different groups of mice. ( c ) Representative TEM images showing morphological changes in the podocyte foot process in different groups of mice. ( d ) Indices for glomerular filtration barrier integrity, including GBM thickness, foot process width and the number of foot processes /μ m GBM (mean±S.E.M.; n =8; * P <0.05 versus control, † P <0.05 versus WT diabetic mice

Article Snippet: Formalin-fixed, paraffin-embedded kidney tissues were sectioned (6- μ m thickness) and assayed for Arrb1 and Arrb2 RNA expression by ISH as described previously., , Arrb2 was detected by the Arrb2 mRNA ISH Kit with digoxigenin (DIG)-labeled probe (Boster Biological Technology Co., Wuhan, China) according to the manufacturer's protocols.

Techniques: Filtration, Control

Journal: Cell Death & Disease

Article Title: β -Arrestins promote podocyte injury by inhibition of autophagy in diabetic nephropathy

doi: 10.1038/cddis.2016.89

Figure Lengend Snippet: β -Arrestin-1 and β -arrestin-2 reduced basal autophagy in podocytes with HG treatment. ( a ) Representative western blotting gel documents and summarized data showing the efficiency of Arrb1 knockdown by shRNA- β -arrestin-1 transfection. ( b ) Representative western blotting gel documents and summarized data showing the efficiency of Arrb2 knockdown by shRNA- β -arrestin-2 transfection. ( c ) Representative electronic micrographs showing autophagosomes in HG-treated podocytes with or without gene silencing of Arrb1 or (and) Arrb2 . The arrows indicate autophagosomes. ( d ) The number of autophagic vesicles (AV) was determined with 10 cells in each sample, respectively. ( e ) Representative western blotting gel documents and summarized data show the levels of LC3-II/LC3-I in the presence of lysosomal inhibitors such as chloroquine (30 μ mol/l) and bafilomycin A1 (50 nmol/l) after Arrb1 knockdown. ( f ) Representative images of LC3 staining by measurement of fluorescent intensity in podocytes in different groups of podocytes infected with RFP–GFP–LC3 adenovirus for 24 h (means±S.E.M.; n =6; * P <0.05 versus control, † P <0.05 versus scramble of HG treatment)

Article Snippet: Formalin-fixed, paraffin-embedded kidney tissues were sectioned (6- μ m thickness) and assayed for Arrb1 and Arrb2 RNA expression by ISH as described previously., , Arrb2 was detected by the Arrb2 mRNA ISH Kit with digoxigenin (DIG)-labeled probe (Boster Biological Technology Co., Wuhan, China) according to the manufacturer's protocols.

Techniques: Western Blot, Knockdown, shRNA, Transfection, Staining, Infection, Control

Journal: Cell Death & Disease

Article Title: β -Arrestins promote podocyte injury by inhibition of autophagy in diabetic nephropathy

doi: 10.1038/cddis.2016.89

Figure Lengend Snippet: β -Arrestin-1 and β -arrestin-2 had not participated in the formation of PI3K core complex. ( a ) Immunoprecipitation assays showing that β -arrestin-1 interacted with VPS34 and beclin-1 and the interaction between VPS34 and β -arrestin-1 was significantly enhanced in podocytes with HG treatment. ( b ) An increased tendency was also observed for the interaction between VPS34 and β -arrestin-2. ( c ) There was no interaction change between beclin-1 and VPS34 in podocytes with HG treatment. ( d ) Gene silencing of Arrb1 or Arrb2 had no effects on the formation of PI3K core complex in podocytes

Article Snippet: Formalin-fixed, paraffin-embedded kidney tissues were sectioned (6- μ m thickness) and assayed for Arrb1 and Arrb2 RNA expression by ISH as described previously., , Arrb2 was detected by the Arrb2 mRNA ISH Kit with digoxigenin (DIG)-labeled probe (Boster Biological Technology Co., Wuhan, China) according to the manufacturer's protocols.

Techniques: Immunoprecipitation

Journal: Cell Death & Disease

Article Title: β -Arrestins promote podocyte injury by inhibition of autophagy in diabetic nephropathy

doi: 10.1038/cddis.2016.89

Figure Lengend Snippet: β -Arrestin-1 and β -arrestin-2 negatively regulated ATG12–ATG5 conjugation system in podocytes with HG treatment. ( a ) Representative western blotting gel documents and summarized data showing the effect of Arrb1 knockdown on the levels of ATG12–ATG5 and LC3-II/LC3-I. ( b ) Representative western blotting gel documents and summarized data showing the effect of Arrb2 knockdown on the levels of ATG12–ATG5 and LC3-II/LC3-I. ( c ) Immunoprecipitation assay showing that β -arrestin-1/2 interacted with ATG7 and the interactions between ATG7 and β -arrestin-1/2 were significantly enhanced in podocytes with HG treatment. (means±S.E.M.; n =6; * P <0.05 versus control, † P <0.05 versus scramble of HG treatment). ( d ) Representative western blotting gel documents and summarized data showing the levels of ATG12–ATG5 and LC3-II/LC3-I in the kidney from Arrb1 −/− diabetic mice. ( e ) Representative western blotting gel documents and summarized data showing the levels of ATG12–ATG5 and LC3-II/LC3-I in the kidney from Arrb2 −/− diabetic mice. Values are means±S.E.M.; * P <0.05 versus control, † P <0.05 versus WT STZ mice ( n =8)

Article Snippet: Formalin-fixed, paraffin-embedded kidney tissues were sectioned (6- μ m thickness) and assayed for Arrb1 and Arrb2 RNA expression by ISH as described previously., , Arrb2 was detected by the Arrb2 mRNA ISH Kit with digoxigenin (DIG)-labeled probe (Boster Biological Technology Co., Wuhan, China) according to the manufacturer's protocols.

Techniques: Conjugation Assay, Western Blot, Knockdown, Immunoprecipitation, Control