Journal: Cell Discovery

Article Title: TAK1 inhibition attenuates both inflammation and fibrosis in experimental pneumoconiosis

doi: 10.1038/celldisc.2017.23

Figure Lengend Snippet: TAK1 as a highly expressed and activated protein in pneumoconiosis. ( a ) Flow chart depicting experimental design in our study. ( b ) Heatmaps showing gene signatures in alveolar macrophage NR8383 cells exposed to silica ( n =3) or vehicle control ( n =3) and lung fibroblast WI-38 cells stimulated with TGF-β ( n =3) or vehicle control ( n =3), respectively. ( c ) Five overlapping genes between top 50 upregulated genes in silica-exposed NR8383 cells and top 50 upregulated genes in TGF-β-stimulated WI-38 cells. ( d ) −ΔΔCT value of mRNA level for the five overlapping genes in lung specimens from pneumoconiosis patients ( n =9) as determined by real-time PCR. GAPDH was used as a control gene for internal correction. Expression of the overlapping genes in control individuals ( n =6) served as the calibrators. ΔΔCT=(CT target −CT GAPDH ) patients −(CT target −CT GAPDH ) controls . ( e ) The level of p-TAK1 in lung specimens from pneumoconiosis patients ( n =9) or control individuals ( n =6) as determined by ELISA (left) and western blot (middle: representative images; right: relative bands intensity). ( f ) Levels of p-TAK1 (T187, T184) and TAK1 were determined by western blotting (left: representative images; right: relative bands intensity) in primary alveolar macrophages isolated from rats with ( n =5) or without silica exposure ( n =5). ( g ) Levels of p-TAK1 (T187, T184) and TAK1 were determined by western blotting (left: representative images; right: relative bands intensity) in primary lung fibroblasts isolated from rats with ( n =5) or without silica exposure ( n =5). Data are presented as mean±s.d. * P <0.05. One-way analysis of variance (ANOVA) with a post hoc test was performed and the statistical differences between the two groups were determined by the Student’s t -test.

Article Snippet: The level of p-TAK1 was quantified using anti-p-TAK1 antibody (Elabscience, Wuhan, China) following established ELISA protocol (TECH TIP no. 65; Thermo Fisher Scientific).

Techniques: Control, Real-time Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Isolation

Journal: Cell Discovery

Article Title: TAK1 inhibition attenuates both inflammation and fibrosis in experimental pneumoconiosis

doi: 10.1038/celldisc.2017.23

Figure Lengend Snippet: Deletion of TAK1 in lung tissues by lentiviral-based CRISPR/Cas9 system reduced inflammation and fibrosis in silica-exposed mice. ( a ) A schematic diagram illustrating the experimental design. Before induction of inflammation and fibrosis in lungs by silica exposure, the mice were infected intratracheally with lentiviral vector expressing CRISPR/Cas9 system (sgTAK1–3 and Cas9) or lentiviral vector control. ( b ) ELISA to determine silica exposure-induced levels of IL-1β (left), TGF-β (middle) and TNF-α (right) in the supernatant of BALF from age-matched mice or mice infected with specified lentiviral vectors. ( c ) Gelatin zymography (upper: representative images; bottom: relative bands intensity) to examine silica exposure-induced activities of matrix metalloproteinases (MMP-9 and MMP-2) in homogenized lung tissues from age-matched mice or mice infected with specified lentiviral vectors. ( d ) MMP-targeted NIR fluorescence imaging showing silica exposure-induced activities of matrix metalloproteinases in lung tissues from age-matched mice or mice infected with specified lentiviral vectors. Scale bar, 1 cm. ( e ) Western blotting (left: representative images; right: relative bands intensity) to examine silica exposure-induced inflammation-related downstream signaling in primary alveolar macrophages isolated from lung tissues of age-matched mice or mice infected with specified lentiviral vectors. ( f ) Hydroxyproline assay to determine silica exposure-induced total collagen levels in lung tissues from age-matched mice or mice infected with specified lentiviral vectors. ( g ) Western blotting (upper: representative images; bottom: relative bands intensity) to determine silica exposure-induced levels of Col I and Col III in lung tissue from age-matched mice or mice infected with specified lentiviral vectors. ( h ) Histological examination to determine silica exposure-induced fibrotic nodule formation (arrows indicated in H&E staining) and collagen deposition (arrows indicated in immunohistochemical staining) in lung tissue from age-matched mice or mice infected with specified lentiviral vectors. Scale bars, 50 μm. ( i ) Western blotting (left: representative images; right: relative bands intensity) to examine silica exposure-induced fibrosis-related downstream signaling in primary fibroblasts isolated from lung tissues of age-matched mice or mice infected with specified lentiviral vectors. Data are presented as mean±s.d. * P <0.05, n =9 per group. One-way ANOVA with a post hoc test was performed and the statistical differences between the two groups were determined by the Student’s t -test.

Article Snippet: The level of p-TAK1 was quantified using anti-p-TAK1 antibody (Elabscience, Wuhan, China) following established ELISA protocol (TECH TIP no. 65; Thermo Fisher Scientific).

Techniques: CRISPR, Infection, Plasmid Preparation, Expressing, Control, Enzyme-linked Immunosorbent Assay, Zymography, Fluorescence, Imaging, Western Blot, Isolation, Hydroxyproline Assay, Staining, Immunohistochemical staining

Journal: Cell Discovery

Article Title: TAK1 inhibition attenuates both inflammation and fibrosis in experimental pneumoconiosis

doi: 10.1038/celldisc.2017.23

Figure Lengend Snippet: Effects of TAK1 gene silencing on inflammation and fibrosis in experimental pneumoconiosis. ( a ) Levels of TAK1 and p-TAK1 were determined by western blotting (left upper: representative images; left bottom: relative bands intensity), inflammatory cytokines (IL-1β, TGF-β and TNF-α) were examined by ELISA (middle) and MMPs (MMP-9 and MMP-2) were analyzed by gelatin zymography (right upper: representative images; right bottom: relative bands intensity) in silica-exposed NR8383 cells with prior incubation of TAK1 siRNA, NC siRNA or vehicle control (Veh: Lipofectamine 2000); n =4 per group. ( b ) Levels of p-TAK1 and TAK1 were determined by western blotting (left upper: representative images; left bottom: relative bands intensity), cell proliferation rate was examined by MTT assay (middle) and collagen subtypes (Col I and Col III) were determined by western blotting (right upper: representative images; right bottom: relative bands intensity) in TGF-β-stimulated WI-38 cells with prior incubation of TAK1 siRNA, NC siRNA or vehicle control (Veh: Lipofectamine 2000). n =4 per group. ( c ) Levels of TAK1 and p-TAK1 were determined by western blotting (left upper: representative images; left bottom: relative bands intensity), inflammatory cytokines (IL-1β, TGF-β and TNF-α) were examined by ELISA (middle) and MMPs (MMP-9 and MMP-2) were analyzed by gelatin zymography (right upper: representative images; right bottom: relative bands intensity) in primary alveolar macrophages with incubation of TAK1 siRNA, NC siRNA or vehicle control (Veh: Lipofectamine 2000). The primary alveolar macrophages were isolated from silica-exposed rats; n =5 per group. ( d ) Levels of p-TAK1 and TAK1 were determined by western blotting (left upper: representative images; left bottom: relative bands intensity), cell proliferation rate was examined by MTT assay (middle) and collagen subtypes (Col I and Col III) were determined by western blotting (right upper: representative images; right bottom: relative bands intensity) in primary lung fibroblasts with incubation of TAK1 siRNA, NC siRNA or vehicle control (Veh: Lipofectamine 2000). The primary lung fibroblasts were isolated from silica-exposed rats; n =5 per group. Data are presented as mean±s.d. * P <0.05. One-way ANOVA with a post hoc test was performed and the statistical differences between the two groups were determined by the Student’s t -test.

Article Snippet: The level of p-TAK1 was quantified using anti-p-TAK1 antibody (Elabscience, Wuhan, China) following established ELISA protocol (TECH TIP no. 65; Thermo Fisher Scientific).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Zymography, Incubation, Control, MTT Assay, Isolation

Journal: Cell Discovery

Article Title: TAK1 inhibition attenuates both inflammation and fibrosis in experimental pneumoconiosis

doi: 10.1038/celldisc.2017.23

Figure Lengend Snippet: In vitro effects of resveratrol on inflammation and fibrosis. ( a ) ELISA to examine levels of IL-1β (left), TGF-β (middle) and TNF-α (right) in the supernatant of alveolar macrophage NR8383 cells with silica exposure and simultaneous administration of vehicle control (Veh: DMSO) or resveratrol (Res) at concentrations of 25, 50 and 100 μ M , respectively. ( b ) Gelatin zymography (upper: representative images; bottom: relative bands intensity) to examine activities of matrix metalloproteinases (MMP-9 and MMP-2) in NR8383 cells with silica exposure and simultaneous administration of vehicle control (Veh: DMSO) or resveratrol (Res) at concentrations of 25, 50 and 100 μ M , respectively. ( c ) Western blotting (upper: representative images; bottom: relative bands intensity) to examine TAK1 activation (p-TAK1) and inflammation-related downstream signaling in NR8383 cells with silica exposure and simultaneous administration of vehicle control (Veh: DMSO) or resveratrol (Res) at concentrations of 25, 50 and 100 μ M , respectively. ( d ) Western blotting (left: representative images; right: relative bands intensity) to determine levels of Col I and Col III in lung fibroblast WI-38 cells with TGF-β-stimulation and simultaneous administration of vehicle control (Veh: DMSO) or resveratrol (Res) at concentrations of 10, 25 and 50 μ M , respectively. ( e ) MTT to examine cell proliferation rate of WI-38 cells with TGF-β-stimulation and simultaneous administration of vehicle control (Veh: DMSO) or resveratrol (Res) at concentrations of 10, 25 and 50 μ M , respectively. ( f ) Western blotting (left: representative images; right: relative bands intensity) to examine TAK1 activation (p-TAK1) and fibrosis-related downstream signaling in WI-38 cells with TGF-β stimulation and simultaneous administration of vehicle control (Veh: DMSO) or resveratrol (Res) at concentrations of 10, 25 and 50 μ M , respectively. NR8383 and WI-38 cells without any treatment were served as blank controls. Data are presented as mean±s.d. * P <0.05, n =4 per group. One-way ANOVA with a post hoc test was performed and the statistical differences between the two groups were determined by the Student’s t -test.

Article Snippet: The level of p-TAK1 was quantified using anti-p-TAK1 antibody (Elabscience, Wuhan, China) following established ELISA protocol (TECH TIP no. 65; Thermo Fisher Scientific).

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Control, Zymography, Western Blot, Activation Assay

Journal: Cell Discovery

Article Title: TAK1 inhibition attenuates both inflammation and fibrosis in experimental pneumoconiosis

doi: 10.1038/celldisc.2017.23

Figure Lengend Snippet: Validation of TAK1 as a molecular target of resveratrol and identification of key residues in TAK1. ( a ) Immunoprecipitation using anti-resveratrol antibody to examine the interaction between TAK1 and resveratrol (Res) in alveolar macrophage NR8383 (left) and lung fibroblasts WI-38 cells (right). TAK1 in the immunoprecipitate and lysate was examined by western blotting (upper: representative images; bottom: relative bands intensity). ( b ) Reference standard of resveratrol (left upper) and immunoprecipitation using anti-TAK1 antibody to examine the interaction between resveratrol and TAK1 in NR8383 (left bottom) and WI-38 cells (right). Resveratrol in the immunoprecipitate was examined by LC-MS/MS. There were two peaks for trans - and cis -resveratrol according to the reference standard. ( c ) Close-up view showing key residues on TAK1 in two predicted binding conformations between TAK1 and resveratrol in molecule docking. ( d ) Immunoprecipitation using anti-resveratrol antibody to examine the interaction between Flag-TAK1 wild-type or Flag-TAK1 mutants (N161R, A107R and N161R/A107R) and resveratrol in NR8383 (left) and WI-38 cells (right). The cells were transfected with pcDNA3.1-based expression vectors for Flag-TAK1 wild-type or Flag-TAK1 mutants (N161R, A107R and N161R/A107R). Flag-TAK1 or Flag-TAK1 mutants in immunoprecipitates and lysates was determined by western blotting (upper: representative images; bottom: relative bands intensity). ( e ) Immunoprecipitation using anti-Flag antibody to examine the interaction between resveratrol and Flag-TAK1 wild-type or Flag-TAK1 mutants in NR8383 cells (upper) and WI-38 cells (bottom). Resveratrol in immunoprecipitate was examined by LC-MS/MS. Experiments were performed at least three times. Res, resveratrol; ND, not detectable.

Article Snippet: The level of p-TAK1 was quantified using anti-p-TAK1 antibody (Elabscience, Wuhan, China) following established ELISA protocol (TECH TIP no. 65; Thermo Fisher Scientific).

Techniques: Biomarker Discovery, Immunoprecipitation, Western Blot, Liquid Chromatography with Mass Spectroscopy, Binding Assay, Transfection, Expressing

Journal: Cell Discovery

Article Title: TAK1 inhibition attenuates both inflammation and fibrosis in experimental pneumoconiosis

doi: 10.1038/celldisc.2017.23

Figure Lengend Snippet: Prevention effects of resveratrol on inflammation and fibrosis in silica-exposed rats. ( a ) A schematic diagram illustrating the experimental design. Briefly, rats were exposed to silica aerosol and simultaneously administered with vehicle (Tween-80) or low-dose resveratrol (10 mg kg −1 ) or high-dose resveratrol (20 mg kg −1 ). After 20 days (4 weeks), the prevention effects of resveratrol on inflammation and related signaling pathway were examined ( b – e ), whereas the prevention effects of resveratrol on fibrosis and related signaling pathway were examined at 40 days (8 weeks) ( f – i ). ( b ) ELISA to determine levels of IL-1β (left), TGF-β (middle) and TNF-α (right) in the supernatant of BALF from rats with silica exposure and simultaneous administration of vehicle control (Veh: Tween-80), low-dose resveratrol (10 mg kg −1 ) or high-dose resveratrol (20 mg kg −1 ). ( c ) Gelatin zymography (upper: representative images; bottom: relative bands intensity) to examine activities of matrix metalloproteinases (MMP-9 and MMP-2) in homogenized lung tissues from rats with silica exposure and simultaneous administration of vehicle control (Veh: Tween-80), low-dose resveratrol (10 mg kg −1 ) or high-dose resveratrol (20 mg kg −1 ). ( d ) MMP-targeted NIR fluorescence imaging showing activities of matrix metalloproteinases in lung tissues from rats with silica exposure and simultaneous administration of vehicle control (Veh: Tween-80), low-dose resveratrol (10 mg kg −1 ) or high-dose resveratrol (20 mg kg −1 ). Scale bar, 1 cm. ( e ) Western blotting (left: representative images; right: relative bands intensity) to examine TAK1 activation (p-TAK1) and inflammation-related downstream signaling in primary alveolar macrophages isolated from lung tissues of rats with silica exposure and simultaneous administration of vehicle control (Veh: Tween-80), low-dose resveratrol (10 mg kg −1 ) or high-dose resveratrol (20 mg kg −1 ). ( f ) Hydroxyproline assay to determine total collagen levels in lung tissues from rats with silica exposure and simultaneous administration of vehicle control (Veh: Tween-80), low-dose resveratrol (10 mg kg −1 ) or high-dose resveratrol (20 mg kg −1 ). ( g ) Western blotting (left: representative images; right: relative bands intensity) to determine levels of Col I and Col III in lung tissue from rats with silica exposure and simultaneous administration of vehicle control (Veh: Tween-80), low-dose resveratrol (10 mg kg −1 ) or high-dose resveratrol (20 mg kg −1 ). ( h ) Histological examination to determine fibrotic nodule formation (arrows indicated in H&E staining) and collagen deposition (arrows indicated in immunohistochemical staining) in lung tissue from rats with silica exposure and simultaneous administration of vehicle control (Veh: Tween-80), low-dose resveratrol (10 mg kg −1 ) or high-dose resveratrol (20 mg kg −1 ). Scale bars, 50 μm. ( i ) Western blotting (left: representative images; right: relative bands intensity) to examine TAK1 activation (p-TAK1) and fibrosis-related downstream signaling in primary fibroblasts isolated from lung tissues of rats with silica exposure and simultaneous administration of vehicle control (Veh: Tween-80), low-dose resveratrol (10 mg kg −1 ) or high-dose resveratrol (20 mg kg −1 ). Rats without any treatment served as a blank control. Data are presented as mean±s.d. * P <0.05, n =9 per group. One-way ANOVA with a post hoc test was performed and the statistical differences between the two groups were determined by the Student’s t -test.

Article Snippet: The level of p-TAK1 was quantified using anti-p-TAK1 antibody (Elabscience, Wuhan, China) following established ELISA protocol (TECH TIP no. 65; Thermo Fisher Scientific).

Techniques: Aerosol, Enzyme-linked Immunosorbent Assay, Control, Zymography, Fluorescence, Imaging, Western Blot, Activation Assay, Isolation, Hydroxyproline Assay, Staining, Immunohistochemical staining

Journal: Cell Discovery

Article Title: TAK1 inhibition attenuates both inflammation and fibrosis in experimental pneumoconiosis

doi: 10.1038/celldisc.2017.23

Figure Lengend Snippet: Intervention effects of resveratrol on inflammation and fibrosis in silica-exposed rats. ( a ) A schematic diagram illustrating the experimental design for the intervention study of resveratrol on inflammatory stage. Briefly, rats were exposed to silica aerosol for 10 days. Then, we administered the rats with resveratrol (20 mg kg −1 ) or vehicle (Tween-80) and simultaneously continued the silica exposure for another 10 days. ( b ) ELISA to determine levels of IL-1β (left), TGF-β (middle) and TNF-α (right) in the supernatant of BALF from silica-exposed rats with the administration of vehicle control (Veh: Tween-80) or resveratrol (20 mg kg −1 ). ( c ) Gelatin zymography (upper: representative images; bottom: relative bands intensity) to examine activities of matrix metalloproteinases (MMP-9 and MMP-2) in homogenized lung tissues from silica-exposed rats with the administration of vehicle control (Veh: Tween-80) or resveratrol (20 mg kg −1 ). ( d ) MMP-targeted NIR fluorescence imaging showing activities of matrix metalloproteinases in silica-exposed rats with the administration of vehicle control (Veh: Tween-80) or resveratrol (20 mg kg −1 ). Scale bar, 1 cm. ( e ) Western blotting (left: representative images; right: relative bands intensity) to examine TAK1 activation (p-TAK1) and inflammation-related downstream signaling in primary alveolar macrophages isolated from lung tissues of silica-exposed rats with treatment of vehicle control (Veh: Tween-80) or resveratrol (20 mg kg −1 ). ( f ) A schematic diagram illustrating the experimental design for the intervention study of resveratrol on fibrotic stage. Briefly, rats were exposed to silica aerosol for 20 days. Then, we administered the rats with 20 mg kg −1 resveratrol or vehicle (Tween-80) and simultaneously continued the silica exposure for another 20 days. ( g ) Hydroxyproline assay to determine levels of total collagen levels in lung tissues from silica-exposed rats with the administration of vehicle control (Veh: Tween-80) or resveratrol (20 mg kg −1 ). ( h ) Western blotting (left: representative images; right: relative bands intensity) to determine levels of Col I and Col III in lung tissue from silica-exposed rats with administration of vehicle control (Veh: Tween-80) or resveratrol (20 mg kg −1 ). ( i ) Histological examination to determine fibrotic nodule formation (arrows indicated in H&E staining) and collagen deposition (arrows indicated in immunohistochemical staining) in lung tissue from silica-exposed rats with treatment of vehicle control (Veh: Tween-80) or resveratrol (20 mg kg −1 ). Rats without any treatment served as blank controls. Scale bars, 50 μm. ( j ) Western blotting (left: representative images; right: relative bands intensity) to examine TAK1 activation (p-TAK1) and fibrosis-related downstream signaling in primary lung fibroblasts isolated from lung tissues of silica-exposed rats with administration of vehicle control (Veh: Tween-80) or resveratrol (20 mg kg −1 ). Data are presented as mean±s.d. * P <0.05, n =9 per group. One-way ANOVA with a post hoc test was performed and the statistical differences between the two groups were determined by the Student’s t -test.

Article Snippet: The level of p-TAK1 was quantified using anti-p-TAK1 antibody (Elabscience, Wuhan, China) following established ELISA protocol (TECH TIP no. 65; Thermo Fisher Scientific).

Techniques: Aerosol, Enzyme-linked Immunosorbent Assay, Control, Zymography, Fluorescence, Imaging, Western Blot, Activation Assay, Isolation, Hydroxyproline Assay, Staining, Immunohistochemical staining

Journal: PLoS ONE

Article Title: A Novel Rabbit Immunospot Array Assay on a Chip Allows for the Rapid Generation of Rabbit Monoclonal Antibodies with High Affinity

doi: 10.1371/journal.pone.0052383

Figure Lengend Snippet: (A) Non-blocking procedure using the TAK1-peptide. Antibodies recognizing the non-phosphorylated peptide (a) and the phosphorylated peptide (b) can bind to biotinylated pTAK1-peptide, and a signal can be detected. (B) Blocking procedure using the TAK1-peptide. Microwell array chips were pre-treated with TAK1-peptide before the addition of biotinylated pTAK1-peptide. a) The TAK1-peptide binds to antibodies that recognize the non-phosphorylated site of the pTAK1-peptide. As a result, the biotinylated pTAK1-peptide cannot bind to the antibodies, and no signal is detected. b) The TAK1-peptide does not bind to antibodies that recognize the phosphorylated site of the pTAK1-peptide. As a result, the biotinylated pTAK1-peptide binds to antibodies, and a signal can be detected. (C) Acquisition efficiency of phosphorylated peptide-specific antibodies in the non-blocking and blocking procedures. The colored pie segment indicates the frequency of RaMoAbs that are specific to a phosphorylated peptide (red) and non-phosphorylated peptide (blue) in the non-blocking procedure (left) and the blocking procedure (right). The number in the center of the pie chart denotes the number of antibodies analyzed. The p -value was determined using Fisher’s test.

Article Snippet: We used hen egg lysozyme (HEL; Sigma), bovine serum albumin (BSA; Wako), human transforming growth factor-β-activated kinase 1 (TAK1)-peptide (TAK1-peptide, DIQTHMNNKGSAA; Operon Biotechnologies), TAK1-peptide phosphorylated at Thr-187 (pTAK1-peptide, DIQTHM(pT)NNKGSAA; Operon Biotechnologies), biotinylated pTAK1-peptide (DIQTHM(pT)NNKGSAAK-biotin; Operon Biotechnologies), and KLH conjugates of pTAK1-peptide (Operon Biotechnologies).

Techniques: Blocking Assay

Journal: PLoS ONE

Article Title: A Novel Rabbit Immunospot Array Assay on a Chip Allows for the Rapid Generation of Rabbit Monoclonal Antibodies with High Affinity

doi: 10.1371/journal.pone.0052383

Figure Lengend Snippet: (A) Examination of the specificity of pTAK1-peptide-specific RaMoAbs. Whole-cell lysates obtained from HeLa cells that were transfected with plasmids containing FLAG-tagged wild type (WT) TAK1 or a phosphorylation site-substituted mutant (T187A) together with HA-tagged TAB1 were separated by SDS-PAGE and immunoblotted with Ra_pTAK01, 04, 05, 06, 14, 19, 21, and 23 antibodies, a commercial antibody specific to phosphorylated TAK1, a TAK1-specific antibody or a TAB1-specific antibody. (B) TNF-α-induced phosphorylation of endogenous TAK1 at Thr187. Whole cell lysates from HeLa cells that had been stimulated with 20 ng ml −1 TNF-α for the indicated time periods were separated by SDS-PAGE and immunoblotted with the Ra_pTAK23 antibody, a commercial pTAK1-pep-specific antibody, or a TAK1-specific antibody.

Article Snippet: We used hen egg lysozyme (HEL; Sigma), bovine serum albumin (BSA; Wako), human transforming growth factor-β-activated kinase 1 (TAK1)-peptide (TAK1-peptide, DIQTHMNNKGSAA; Operon Biotechnologies), TAK1-peptide phosphorylated at Thr-187 (pTAK1-peptide, DIQTHM(pT)NNKGSAA; Operon Biotechnologies), biotinylated pTAK1-peptide (DIQTHM(pT)NNKGSAAK-biotin; Operon Biotechnologies), and KLH conjugates of pTAK1-peptide (Operon Biotechnologies).

Techniques: Transfection, Mutagenesis, SDS Page