Journal: Nature Communications

Article Title: Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M

doi: 10.1038/ncomms7062

Figure Lengend Snippet: ( a ) IRAK-M mRNA expression in human bronchial epithelial BEAS-2B cells treated with DEX (0.01, 0.1, 1, 10, 100 or 1,000 nM) for 1 h, followed by the stimulation of NTHi for 5 h. ( b ) IRAK-M mRNA expression in human lung epithelial A549 cells stimulated with DEX (100 nM) for 1 h, followed by NTHi for indicated time. ( c ) A549 cells were treated with DEX (100 nM) for 1 h, followed by NTHi for 12 h. IRAK-M protein was detected by anti-IRAK-M antibody. ( d ) IRAK-M protein expression was quantified from three independent experiments. ( e ) IRAK-M mRNA expression in primary NHBE cells treated with DEX (10, 100 or 1,000 nM) for 1 h and subjected to NTHi stimulation for 5 h. ( f ) Human peripheral blood CD14 + monocytes were differentiated to macrophages by culturing them with granulocyte–macrophage colony-stimulating factor for 7 days. IRAK-M mRNA was determined after the macrophages were treated with DEX (100 nM) for 1 h, followed by stimulation of NTHi for 5 h. ( g ) Mice were stimulated with DEX (1 mg kg −1 , intraperitoneally) for 2 h and intratracheally inoculated with NTHi (1 × 10 7 c.f.u.) for 24 h. The mRNA expression in alveolar macrophages was assessed by qPCR. ( h ) The IRAK-M mRNA expression in lung was assessed by qPCR. ( i ) Immunostaining of mouse lung with control IgG or anti-IRAK-M antibody by LSAB (Labelled Streptavidin Biotin) staining system (Scale bar, 50 μm; magnification, × 400). ( j ) Treatment-blind observers scored the IRAK-M expression from the histology results. ( k ) The lung sections were stained by using indicated antibodies. The arrows and arrowheads indicate the epithelial cells and macrophages merged with IRAK-M expression, respectively. (Scale bar, 50 μm; magnification, × 400. Data in a , b , d - h , n =3; j , n =6) are mean±s.d. * P< 0.05; t- test.

Article Snippet: The detection of IRAK-M protein was performed using rabbit anti-IRAK-M (ProSci, 2 μg ml −1 for Immunohistochemistry, 10 μg ml −1 for Immunofluorescence) and Donkey anti-rabbit-FITC (Santa Cruz Biotechnology, 5 μg ml −1 ) in the paraffin section of mouse lung tissue.

Techniques: Expressing, Immunostaining, Staining

Journal: Nature Communications

Article Title: Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M

doi: 10.1038/ncomms7062

Figure Lengend Snippet: ( a ) The proteins from BEAS-2B cells stably overexpressing IRAK-M and its control pcDNA3.1 (Mock) cells were detected by immunoblot with indicated antibodies. ( b ) The mRNA expression was detected by qPCR after the stable cells were treated with DEX (100 nM) and NTHi. ( c ) BEAS-2B cells were transfected with siRNA control or IRAK-M for 72 h. IRAK-M protein expression was detected by immunoblot with anti-IRAK-M antibodies (SC, Santa Cruz Biotechnology sc-100389; PS, ProSci #2355). ( d ) siRNA-transfected BEAS-2B cells were infected with NTHi followed by qPCR. ( e ) The mRNA level was calculated by following: (NTHi-induced mRNA at each concentration of DEX)/(NTHi-induced mRNA without DEX). Data in b , d , e , n =3) are mean±s.d. * P< 0.05; t- test.

Article Snippet: The detection of IRAK-M protein was performed using rabbit anti-IRAK-M (ProSci, 2 μg ml −1 for Immunohistochemistry, 10 μg ml −1 for Immunofluorescence) and Donkey anti-rabbit-FITC (Santa Cruz Biotechnology, 5 μg ml −1 ) in the paraffin section of mouse lung tissue.

Techniques: Stable Transfection, Western Blot, Expressing, Transfection, Infection, Concentration Assay

Journal: Nature Communications

Article Title: Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M

doi: 10.1038/ncomms7062

Figure Lengend Snippet: WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with NTHi (1 × 10 7 c.f.u. per lung) for 24 h. ( a ) The mRNA expression in the mouse lung tissue was analysed by qPCR. ( b ) The protein level of proinflammatory mediators in BAL fluid was determined by enzyme-linked immunosorbent assay. ( c ) The mRNA expression in alveolar macrophages was analysed by qPCR. Data in a , c , n =3; b , n =9 are mean±s.d. * P< 0.05, NS, non-significant; t- test.

Article Snippet: The detection of IRAK-M protein was performed using rabbit anti-IRAK-M (ProSci, 2 μg ml −1 for Immunohistochemistry, 10 μg ml −1 for Immunofluorescence) and Donkey anti-rabbit-FITC (Santa Cruz Biotechnology, 5 μg ml −1 ) in the paraffin section of mouse lung tissue.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M

doi: 10.1038/ncomms7062

Figure Lengend Snippet: ( a – e ) WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with NTHi (1 × 10 7 c.f.u. per lung) for 24 h. ( a ) Haematoxylin and eosin (H&E) staining of lung tissues from mice (Scale bar, 200 μm, magnification, × 100 in large frame; Scale bar 50 μm, magnification, × 400 in inserted frame). ( b ) Blinded histopathologic scoring of lung inflammation was performed on H&E-stained lung sections in a grade 0–3. ( c ) The number of PMN cells from BAL fluid was counted using a haemocytometer under the microscope. ( d ) Bacterial loads (c.f.u.) in lung homogenates. ( e ) The mRNA expression of mBD4 in lung was measured by qPCR ( f ) WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with lethal dose of NTHi (5 × 10 8 c.f.u. per lung) for 7 days. Survival rate was monitored for indicated days. P values were determined by Kaplan–Meier survival analysis with GraphPad Prism 5.0. Data in b =8; c , e , n =3; d =10, f =20 are mean±s.d. * P< 0.05, NS, non-significant; t- test.

Article Snippet: The detection of IRAK-M protein was performed using rabbit anti-IRAK-M (ProSci, 2 μg ml −1 for Immunohistochemistry, 10 μg ml −1 for Immunofluorescence) and Donkey anti-rabbit-FITC (Santa Cruz Biotechnology, 5 μg ml −1 ) in the paraffin section of mouse lung tissue.

Techniques: Staining, Microscopy, Expressing

Journal: Nature Communications

Article Title: Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M

doi: 10.1038/ncomms7062

Figure Lengend Snippet: ( a ) Immunoblot shows that IRAK-M protein expression in BEAS-2B cells treated with DEX (100 nM) and NTHi, TNF-α (10 ng ml −1 ), IL-1β (1 ng ml −1 ) or Pam3CSK4 (1 μg ml −1 ). ( b ) IRAK-M mRNA expression assessed by qPCR in A549 cells transfected with constitutive active form of IKKβ (IKKβ CA) with DEX (100 nM). ( c – f ), IRAK-M expression in BEAS-2B cells treated with DEX (100 nM) and NTHi, IKKβ inhibitor (1 μM) ( c , d ) or RU486 (1 μM; e , f ). ( g ) Immunoblot shows IRAK-M protein expression in BEAS-2B cells transfected with siRNA GR. ( h ) BEAS-2B cells were treated with NTHi, DEX (100 nM) and actinomycin D, ActD (5 μg ml −1 ), followed by immunoblot. ( i ) The expression of IRAK-M mRNA in BEAS-2B cells treated with DEX (100 nM) and Compound A, CpdA (1 or 10 μM) followed by NTHi stimulation for 5 h. ( j ) Relative luciferase activity (RLA) of NF-κB is measured by luciferase assay. BEAS-2B cells transfected with NF-κB luciferase plasmid were treated with CpdA and NTHi. Data ( b , c , e , i , j , n =3) are mean±s.d. * P< 0.05, NS; t- test.

Article Snippet: The detection of IRAK-M protein was performed using rabbit anti-IRAK-M (ProSci, 2 μg ml −1 for Immunohistochemistry, 10 μg ml −1 for Immunofluorescence) and Donkey anti-rabbit-FITC (Santa Cruz Biotechnology, 5 μg ml −1 ) in the paraffin section of mouse lung tissue.

Techniques: Western Blot, Expressing, Transfection, Luciferase, Activity Assay, Plasmid Preparation

Journal: Nature Communications

Article Title: Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M

doi: 10.1038/ncomms7062

Figure Lengend Snippet: ( a – c ) Relative luciferase activity (RLA) was measured after the transfection of IRAK-M promoter in pGL3 basic vector and treated with DEX (100 nM) and NTHi in BEAS-2B cells ( a , b ). ( c ) MEF cells were transfected with IRAK-M-luc (−500 bp), followed by DEX (1 μM) and NTHi stimulation. ( d ) Immunoblot shows IRAK-M protein expression in BEAS-2B cells transfected with siRNA p65. ( e , f ) ChIP assay in BEAS-2B cells treated with DEX and NTHi for 1 h. IRAK-M promoter regions from −38 to +39 bp for GRE, from −231 to −99 bp for κB site were amplified by qPCR. ( g ) Re-ChIP assay (detailed in Methods). PCR products were detected in agarose gel. ( h , i ) ChIP assay in BEAS-2B cells treated with IKKβ inhibitor (1 μM), RU486 (1 μM), DEX (100 nM) and NTHi. Data ( a – c , n =3; e , f , n =2) are mean±s.d. * P<0.05 ; t- test.

Article Snippet: The detection of IRAK-M protein was performed using rabbit anti-IRAK-M (ProSci, 2 μg ml −1 for Immunohistochemistry, 10 μg ml −1 for Immunofluorescence) and Donkey anti-rabbit-FITC (Santa Cruz Biotechnology, 5 μg ml −1 ) in the paraffin section of mouse lung tissue.

Techniques: Luciferase, Activity Assay, Transfection, Plasmid Preparation, Western Blot, Expressing, Amplification, Agarose Gel Electrophoresis

Journal: Nature Communications

Article Title: Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M

doi: 10.1038/ncomms7062

Figure Lengend Snippet: A schematic representation of suppression of inflammation by GCs via the upregulation of IRAK-M expression.

Article Snippet: The detection of IRAK-M protein was performed using rabbit anti-IRAK-M (ProSci, 2 μg ml −1 for Immunohistochemistry, 10 μg ml −1 for Immunofluorescence) and Donkey anti-rabbit-FITC (Santa Cruz Biotechnology, 5 μg ml −1 ) in the paraffin section of mouse lung tissue.

Techniques: Expressing