Journal: Nature Communications
Article Title: SENP1-mediated NEMO deSUMOylation in adipocytes limits inflammatory responses and type-1 diabetes progression
Figure Lengend Snippet: SENP1 deletion alters pancreatic adipocyte phenotype and augments NF-κB-dependent inflammation. ( a , b ) Pancreatic adipose was collected from 7 and 14 weeks old Ctrl and SENP1-aP2KO male (male, n =6). Morphology was visualized by haematoxylin and eosin stain staining. Scale bar, 20 μm ( a ). Cell sizes were quantified in ( b ). Three sections from each adipose tissue. Data are representative for three independent experiments. ( c ) Transcript levels of adipocyte differentiation markers (fatty acid synthase, adipose triglyceride lipase and lipoprotein lipase) in PATs were quantified by quantitative PCR with reverse transcription with GAPDH for normalization. n =6, male for each group. ( d ) Increased IKK-NF-κB activities in SENP1-aP2KO PATs. A representative western blot was from Ctrl and SENP1-aP2KO mice at the age of 7 weeks. Data are representative from three independent experiments is shown ( n =6, male). Relative protein levels were quantified from three blots by taking Ctrl as 1.0 ( n =6, male). ( e – h ) NF-κB activation is specifically detected in PATs of SENP1-aP2KO mice. ( e ) Phosphor-p65 staining (red) in PATs but not in pancreas. ( f ) Co-immunostaining of phosphor-p65 (green) and adipocyte marker FABP4 (red). ( g ) High-power images show co-staining of phosphor-p65 (red) in the nucleus of FABP4 + adipocytes (green). ( h ) Co-staining of phosphor-p65 (green) with APC-conjugated adipocyte marker FABP4 (green; yellow arrowheads), but not with macrophage marker F4/80 (red; white arrowheads). Representative images are from one of three sections from SENP1-aP2KO PATs and n =3 male mice. Scale bar, 20 μm. ( i , j ) ChIP assay. ChIP assays with p65/RelA antibody were performed in the adipocyte isolated from Ctrl ( n =6, male) and SENP1-aP2KO mice ( n =6, male) with an IgG isotype as a control. Bindings of p65/RelA to the IL-6, IL-1β and TNF-α gene promoters were quantified with the ratio of IP/input for each promoter ( j ). ( k , l ) Effects of SENP1 knockdown on NF-κB and cytokine expression in adipocytes. 3T3-L1 adipocytes were transfected with control or SENP1-specific siRNA for 24 h. IKK-NF-κB p65/RelA signalling molecules were detected by western blotting. A representative blot from three experiments is shown and protein levels are quantified by taking Ctrl as 1.0 ( k ). Protein levels of IL-6, TNF-α, IFN-γ and IL-1β levels present in adipocyte cultures were detected with ELISA after 24-h culture ( l ). All data are means±s.e.m. of three independent experiments. The two-tailed Student's paired t -test was used for the statistical analysis. * P
Article Snippet: Cell lines and isolation of mature adipocytes 3T3-L1, a commonly used preadipocyte mouse cell line, was obtained from ATCC (CL-173) and cultured under DMEM media supplemented with 2 mM glutamine and 10% calf serum.
Techniques: H&E Stain, Staining, Real-time Polymerase Chain Reaction, Western Blot, Mouse Assay, Activation Assay, Immunostaining, Marker, Chromatin Immunoprecipitation, Isolation, Expressing, Transfection, Enzyme-linked Immunosorbent Assay, Two Tailed Test