Journal: Cell Death & Disease

Article Title: IRAK1 mediates TLR4-induced ABCA1 downregulation and lipid accumulation in VSMCs

doi: 10.1038/cddis.2015.212

Figure Lengend Snippet: IRAK1 was involved in the effects of TLR4 signaling on VSMCs lipid accumulation and ABCA1 expression in vitro . WT and TLR4 −/− VSMCs were incubated with or without 50 μ g/ml oxLDL for 24, 48 and 72 h. ( a and b ) Western blot was used to show IRAK1 and p-IRAK1 protein expression in oxLDL-treated or untreated VSMCs. Increased expression of p-IRAK1 protein was detected in oxLDL-treated WT VSMCs ( a , n =3 experiments in duplicate), but not in TLR4 −/− VSMCs ( b , n =3 experiments in duplicate). ( c ) IRAK1 kinase activity was significantly elevated in WT VSMCs stimulated with oxLDL, but significantly inhibited in TLR4 −/− VSMCs in response to oxLDL, as determined by an in vitro kinase assay ( n =2 experiments in duplicate). ( d – f ) WT VSMCs were transfected with IRAK1-specific siRNA using a transfection reagent. A non-related scrambled siRNA was used as a negative control (NC-siRNA). At 24 h following transfection, the cells were exposed to 50 μ g/ml oxLDL for 48 h. IRAK1 deficiency upregulated ABCA1 expression ( d , n =3 experiments in duplicate) and attenuated lipid accumulation in WT VSMCs as determined by BODIPY 493/503 staining ( e , green fluorescence, scale bar=20 μ m). Quantification of lipid accumulation was based on the OD values for destained Oil Red O in WT VSMCs ( f , n =4 experiments in duplicate). All data are represented as the fold change relative to the controls and are expressed as the mean±S.E.M. * P <0.05, ** P <0.01 compared with the untreated control group

Article Snippet: Following this, the membranes were incubated with primary antibodies against TLR4, ABCA1 (Abcam, Burlingame, CA, USA), IRAK1 (Santa Cruz Biotechnology) or p-IRAK1 (phospho-Thr387, Biorbyt, Cambridge, UK).

Techniques: Expressing, In Vitro, Incubation, Western Blot, Activity Assay, Kinase Assay, Transfection, Negative Control, Staining, Fluorescence, Control

Journal: Cell Death & Disease

Article Title: IRAK1 mediates TLR4-induced ABCA1 downregulation and lipid accumulation in VSMCs

doi: 10.1038/cddis.2015.212

Figure Lengend Snippet: Effects of TLR4 signaling induced by a high-fat diet (HFD) on IRAK1 activity and ABCA1 expression in vivo . WT mice were either fed a normal chow diet (NCD) or a very high-fat diet for 12 weeks and then their thoracic aortas were harvested. ( a ) Hematoxylin and eosin (HE) staining on cross-sections from thoracic aorta were presented. Artery sections were double-stained with TLR4 (green fluorescence) and p-IRAK1 (red fluorescence) antibodies and co-localization was determined in the merged images. ( b ) Immunohistochemical studies were performed by co-staining TLR4 (green fluorescence) and ABCA1 (red fluorescence). The merged panel indicates the co-localization of TLR4 with ABCA1. In images a and b , the nuclei were stained with DAPI (blue fluorescence). Scale bar in HE images=100 μ m; scale bar in immunofluorescence images=20 μ m. ( c and d ) The p-IRAK1 or ABCA1 fluorescence intensity mean values were determined by the mean±S.E.M. of n =6 non-consecutive sections from five mice. ** P <0.01

Article Snippet: Following this, the membranes were incubated with primary antibodies against TLR4, ABCA1 (Abcam, Burlingame, CA, USA), IRAK1 (Santa Cruz Biotechnology) or p-IRAK1 (phospho-Thr387, Biorbyt, Cambridge, UK).

Techniques: Activity Assay, Expressing, In Vivo, Staining, Fluorescence, Immunohistochemical staining, Immunofluorescence

Journal: Cell Death & Disease

Article Title: IRAK1 mediates TLR4-induced ABCA1 downregulation and lipid accumulation in VSMCs

doi: 10.1038/cddis.2015.212

Figure Lengend Snippet: In vivo TLR4 knockout inhibited high-fat diet (HFD)-induced IRAK1 activation and rescued ABCA1 downregulation. ( a and b ) TLR4 −/− mice were fed either a normal chow diet (NCD) or a very HFD for 12 weeks. Following this, their thoracic aortas were obtained. Thoracic aortas were sectioned, and stained for hematoxylin and eosin. IRAK1 activation (red fluorescence) and ABCA1 expression (red fluorescence) were determined using immunofluorescence staining. DAPI was used for nuclear counterstaining (blue fluorescence). ( c and d ) Determination of p-IRAK1 and ABCA1 fluorescence intensity mean values ( n =6 non-consecutive sections from 5 mice). ( e – g ) WT and TLR4 −/− mice were fed either a normalized chow diet or a very HFD for 12 weeks. Following this, aortic root sections were obtained. A negative control was performed using phosphate-buffered saline (PBS) instead of primary antibodies. BF (bright field) images show cross-sections of the arteries from which the immunofluorescence images were obtained ( e ). Detection of lipid accumulation in aortic roots using BODIPY 493/503 staining (green fluorescence). Significant lipid accumulation was observed in aortic root sections from WT mice fed a HFD ( f ), but not from TLR4 −/− mice fed a HFD ( g ). The white arrows show representative lipid accumulation. ( h ) Determination of fluorescence (normalized to plaque area, n =6 non-consecutive sections from 5 mice). Scale bar in BF and HE images=100 μ m; scale bar in immunofluorescence images=20 μ m. Data are expressed as the mean±S.E.M. ** P <0.01 compared with NCD group

Article Snippet: Following this, the membranes were incubated with primary antibodies against TLR4, ABCA1 (Abcam, Burlingame, CA, USA), IRAK1 (Santa Cruz Biotechnology) or p-IRAK1 (phospho-Thr387, Biorbyt, Cambridge, UK).

Techniques: In Vivo, Knock-Out, Activation Assay, Staining, Fluorescence, Expressing, Immunofluorescence, Negative Control, Saline