Journal: Respiratory Research

Article Title: Role of IGF-1 pathway in lung fibroblast activation

doi: 10.1186/1465-9921-14-102

Figure Lengend Snippet: Decreased αSMA promoter activity and αSMA protein expression in bleomycin-injured mice treated with IGF-1 receptor blocking antibody (A12). (A) Representative H&E sections of αSMA-GFP mice at day 21 after bleomycin injury. Fibrotic regions of lung parenchyma are indicated by ( ) and normal lung parenchyma are indicated by ( n ). (B) αSMA immunostaining of bleomycin-injured lung in an αSMA-GFP mouse. Note the overlap of αSMA staining (red) with αSMA-GFP expression (green) in the peribronchiolar fibrotic region indicated by ( ). Scale bars represent 100 μm. (C) (Left) Representative fluorescent images of αSMA-GFP mice treated with A12 (b and d) showed less αSMA promoter activity as indicated by GFP (green) positive cells, compared to control mice (a and c) at d21 after bleomycin instillation. (Right) Percentage of αSMA-GFP + cells/total number of DAPI + cells, quantification by NIH ImageJ (n = 4 mice/group, mean ± SEM). (D) (Left) Representative images of αSMA staining by immunofluorescent microscopy of the same A12-treated mice (b and d) compared to control mice (a and c) at d21 after bleomycin instillation. Large airways and vasculature staining for αSMA, indicated by an asterisk (*), were masked in the analysis. Interstitial staining, indicated by an arrow ( ), was included in the analysis. (Right) Ratio of αSMA staining area per DAPI + area (n = 4 mice/group, mean ± SEM).

Article Snippet: Function-blocking antibody to the human IGF-1 receptor (A12) and keyhole limpet hemocyanin (KLH) isotype control antibody were a generous gift from Dale Ludwig (ImClone Systems) [ , ].

Techniques: Activity Assay, Expressing, Blocking Assay, Immunostaining, Staining, Control, Microscopy

Journal: Respiratory Research

Article Title: Role of IGF-1 pathway in lung fibroblast activation

doi: 10.1186/1465-9921-14-102

Figure Lengend Snippet: Effect of matrix stiffness on response to IGF-1 treatment. (A) MLF on tissue culture plate or collagen I-coated tissue culture plate (stiff substrates) were treated with IGF-1 (100 ng/ml), TGFβ (1 ng/ml) or IGF/TGFβ (100 ng/ml and 1 ng/ml, respectively), or serum-free media (negative control) for 24 hr. (B) MLF on collagen I (1 mg/ml) hydrogel (soft substrate) were treated with IGF-1 (100 ng/ml), IGF-1 (100 ng/ml) with A12 (40 μg/ml) or PI3 kinase inhibitor LY294002 (Ly, 50 μM;) for 24 h. (C) MLF isolated from bleomycin-injured C57Bl6 mice were treated with the indicated cytokine. Real time PCR analyses of myofibroblast markers Acta2 , Col1a1 , and Col3a1 were performed. Data were normalized to HPRT expression. Y-axis represents fold increase compared to serum-free control (n = 3, mean ± SEM, * p < 0.05 compared to serum-free control).

Article Snippet: Function-blocking antibody to the human IGF-1 receptor (A12) and keyhole limpet hemocyanin (KLH) isotype control antibody were a generous gift from Dale Ludwig (ImClone Systems) [ , ].

Techniques: Negative Control, Isolation, Real-time Polymerase Chain Reaction, Expressing, Control

Journal: Respiratory Research

Article Title: Role of IGF-1 pathway in lung fibroblast activation

doi: 10.1186/1465-9921-14-102

Figure Lengend Snippet: IGF-1 treatment increases αSMA stress fibers. A . MLF treated with IGF-1 (100 ng/ml), TGF-β1 (10 ng/ml) or IGF-1/TGF-β1 (100 ng/ml and 10 ng/ml, respectively) with or without A12 (40 μg/ml) for 24 hr. Negative control is serum free media. Cells were co-stained for F-actin (red) and αSMA (green). B . The mean ratio (±SEM) of αSMA stress fiber (+) fibroblasts over all F-actin (+) fibroblasts is presented in the bar graph. * p < 0.05 compared to serum-free control. ** p < <0.01 compared to serum-free control.

Article Snippet: Function-blocking antibody to the human IGF-1 receptor (A12) and keyhole limpet hemocyanin (KLH) isotype control antibody were a generous gift from Dale Ludwig (ImClone Systems) [ , ].

Techniques: Negative Control, Staining, Control

Journal: Animal Nutrition

Article Title: Cytochrome P450 enzymes mediated by DNA methylation is involved in deoxynivalenol-induced hepatoxicity in piglets

doi: 10.1016/j.aninu.2021.11.009

Figure Lengend Snippet: Liver functional indicators, CYP450 enzyme, DNA methyltransferase expression and genome-wide 5-mC level in the liver of piglets fed diets containing DON-contaminated corn ( n = 7). (A) Change of serum liver functional indicators AST, ALT and GGT. (B and C) The mRNA levels of CYP1A1, CYP1A2, CYP2B22, CYP2C33, CYP2D25, CYP2E1, CYP3A22, CYP3A29 and DNMT1, DNMT3A and DNMT3B were detected by qPCR. (D) The 5-mC level of genomic genes was assessed by the colorimetric. (E) The protein levels of CYP1A1, CYP2E1 and CYP3A29 were detected by Western blot. ∗ P < 0.05, ∗∗ P < 0.01 versus 0 mg/kg group. ALT = alanine aminotransferase; AST = aspartate aminotransferase; CYP = cytochrome P; DNMT = DNA methyltransferases; GGT = glutamyl transpeptidase; 5-mC = 5-methyl cytosine.

Article Snippet: Blots were blocked with 5% bovine serum albumin (BSA) for 1 h and incubated with primary antibodies (species specificity: Human, Mouse, Rat) (ABclonal Technology, Wuhan, China) for NNMT Rabbit pAb (A18033) (1:500), CYP1A1 Rabbit pAb (A2159) (1:1,000), CYP2E1 Rabbit pAb (A2160) (1:1,000), CYP3A4 Rabbit pAb (1:1,000), IGF1 Rabbit pAb (A11985) (1:500), GALP Rabbit pAb (A17445) (1:1,000) and GAPDH Rabbit mAb (A19056) (1:2,000) dilution overnight at 4 °C.

Techniques: Functional Assay, Expressing, Genome Wide, Western Blot

Journal: Animal Nutrition

Article Title: Cytochrome P450 enzymes mediated by DNA methylation is involved in deoxynivalenol-induced hepatoxicity in piglets

doi: 10.1016/j.aninu.2021.11.009

Figure Lengend Snippet: The effects of different concentrations of DON on the expression of CYP450 enzymes and growth-related genes, on cell damage and cell growth were tested in human normal hepatocytes L02 cells. (A) The mRNA levels of CYP1A1, CYP2E1, CYP3A4, NNMT, IGF-1 and IGFBP2 were detected by qPCR. (B and C) The protein levels of CYP1A1, CYP2E1, CYP3A4, and IGF-1 were detected by Western blot. (D) The pathological changes of L02 cells under DON exposure were detected by H&E staining. (E) L02 cell proliferation after DON treatment was detected by the EdU reagent (100 × magnification, scale bar = 50 μm). ∗ P < 0.05, ∗∗ P < 0.01 versus the control group. EdU = 5-ethynyl-2′-deoxyuridine.

Article Snippet: Blots were blocked with 5% bovine serum albumin (BSA) for 1 h and incubated with primary antibodies (species specificity: Human, Mouse, Rat) (ABclonal Technology, Wuhan, China) for NNMT Rabbit pAb (A18033) (1:500), CYP1A1 Rabbit pAb (A2159) (1:1,000), CYP2E1 Rabbit pAb (A2160) (1:1,000), CYP3A4 Rabbit pAb (1:1,000), IGF1 Rabbit pAb (A11985) (1:500), GALP Rabbit pAb (A17445) (1:1,000) and GAPDH Rabbit mAb (A19056) (1:2,000) dilution overnight at 4 °C.

Techniques: Expressing, Western Blot, Staining, Control

Journal: Animal Nutrition

Article Title: Cytochrome P450 enzymes mediated by DNA methylation is involved in deoxynivalenol-induced hepatoxicity in piglets

doi: 10.1016/j.aninu.2021.11.009

Figure Lengend Snippet: Effects of CYP2E1 silencing on cell proliferation. After L02 cells were transfected with the siNC and siCYP2E1 for 24 h, and then added DON (5 μmol/L) for 24 h. (A and B) The expression levels of CYP2E1 and IGF-1 were analyzed by qPCR and Western blot methods. (C) L02 cell proliferation was detected by the EdU reagent (100 × magnification, scale bar = 50 μm). (D) The level of cell proliferation in Fig. 6C was quantified. ∗ P < 0.05, ∗∗ P < 0.01 versus the siNC group; # P < 0.05, ## P < 0.01 versus the siNC + DON group, and the data were expressed as SD ( n = 3).

Article Snippet: Blots were blocked with 5% bovine serum albumin (BSA) for 1 h and incubated with primary antibodies (species specificity: Human, Mouse, Rat) (ABclonal Technology, Wuhan, China) for NNMT Rabbit pAb (A18033) (1:500), CYP1A1 Rabbit pAb (A2159) (1:1,000), CYP2E1 Rabbit pAb (A2160) (1:1,000), CYP3A4 Rabbit pAb (1:1,000), IGF1 Rabbit pAb (A11985) (1:500), GALP Rabbit pAb (A17445) (1:1,000) and GAPDH Rabbit mAb (A19056) (1:2,000) dilution overnight at 4 °C.

Techniques: Transfection, Expressing, Western Blot

Journal: Animal Nutrition

Article Title: Cytochrome P450 enzymes mediated by DNA methylation is involved in deoxynivalenol-induced hepatoxicity in piglets

doi: 10.1016/j.aninu.2021.11.009

Figure Lengend Snippet: Liver functional indicators, CYP450 enzyme, DNA methyltransferase expression and genome-wide 5-mC level in the liver of piglets fed diets containing DON-contaminated corn ( n = 7). (A) Change of serum liver functional indicators AST, ALT and GGT. (B and C) The mRNA levels of CYP1A1, CYP1A2, CYP2B22, CYP2C33, CYP2D25, CYP2E1, CYP3A22, CYP3A29 and DNMT1, DNMT3A and DNMT3B were detected by qPCR. (D) The 5-mC level of genomic genes was assessed by the colorimetric. (E) The protein levels of CYP1A1, CYP2E1 and CYP3A29 were detected by Western blot. ∗ P < 0.05, ∗∗ P < 0.01 versus 0 mg/kg group. ALT = alanine aminotransferase; AST = aspartate aminotransferase; CYP = cytochrome P; DNMT = DNA methyltransferases; GGT = glutamyl transpeptidase; 5-mC = 5-methyl cytosine.

Article Snippet: Blots were blocked with 5% bovine serum albumin (BSA) for 1 h and incubated with primary antibodies (species specificity: Human, Mouse, Rat) (ABclonal Technology, Wuhan, China) for NNMT Rabbit pAb (A18033) (1:500), CYP1A1 Rabbit pAb (A2159) (1:1,000), CYP2E1 Rabbit pAb (A2160) (1:1,000), CYP3A4 Rabbit pAb (1:1,000), IGF1 Rabbit pAb (A11985) (1:500), GALP Rabbit pAb (A17445) (1:1,000) and GAPDH Rabbit mAb (A19056) (1:2,000) dilution overnight at 4 °C.

Techniques: Functional Assay, Expressing, Genome Wide, Western Blot

Journal: Animal Nutrition

Article Title: Cytochrome P450 enzymes mediated by DNA methylation is involved in deoxynivalenol-induced hepatoxicity in piglets

doi: 10.1016/j.aninu.2021.11.009

Figure Lengend Snippet: The effects of different concentrations of DON on the expression of CYP450 enzymes and growth-related genes, on cell damage and cell growth were tested in human normal hepatocytes L02 cells. (A) The mRNA levels of CYP1A1, CYP2E1, CYP3A4, NNMT, IGF-1 and IGFBP2 were detected by qPCR. (B and C) The protein levels of CYP1A1, CYP2E1, CYP3A4, and IGF-1 were detected by Western blot. (D) The pathological changes of L02 cells under DON exposure were detected by H&E staining. (E) L02 cell proliferation after DON treatment was detected by the EdU reagent (100 × magnification, scale bar = 50 μm). ∗ P < 0.05, ∗∗ P < 0.01 versus the control group. EdU = 5-ethynyl-2′-deoxyuridine.

Article Snippet: Blots were blocked with 5% bovine serum albumin (BSA) for 1 h and incubated with primary antibodies (species specificity: Human, Mouse, Rat) (ABclonal Technology, Wuhan, China) for NNMT Rabbit pAb (A18033) (1:500), CYP1A1 Rabbit pAb (A2159) (1:1,000), CYP2E1 Rabbit pAb (A2160) (1:1,000), CYP3A4 Rabbit pAb (1:1,000), IGF1 Rabbit pAb (A11985) (1:500), GALP Rabbit pAb (A17445) (1:1,000) and GAPDH Rabbit mAb (A19056) (1:2,000) dilution overnight at 4 °C.

Techniques: Expressing, Western Blot, Staining, Control

Journal: Animal Nutrition

Article Title: Cytochrome P450 enzymes mediated by DNA methylation is involved in deoxynivalenol-induced hepatoxicity in piglets

doi: 10.1016/j.aninu.2021.11.009

Figure Lengend Snippet: Effects of CYP1A1 silencing on cell proliferation. After L02 cells were transfected with the siNC and siCYP1A1 for 24 h, and then added DON (5 μmol/L) for 24 h. (A and B) The expression levels of CYP1A1 and IGF-1 were detected by qPCR and Western blot. (C) L02 cell proliferation was detected by the EdU reagent (100 × magnification, scale bar = 50 μm). (D) The level of cell proliferation in Fig. 5C was quantified. ∗ P < 0.05, ∗∗ P < 0.01 versus the siNC group; # P < 0.05, ## P < 0.01 versus the siNC + DON group, and the data were expressed as SD ( n = 3).

Article Snippet: Blots were blocked with 5% bovine serum albumin (BSA) for 1 h and incubated with primary antibodies (species specificity: Human, Mouse, Rat) (ABclonal Technology, Wuhan, China) for NNMT Rabbit pAb (A18033) (1:500), CYP1A1 Rabbit pAb (A2159) (1:1,000), CYP2E1 Rabbit pAb (A2160) (1:1,000), CYP3A4 Rabbit pAb (1:1,000), IGF1 Rabbit pAb (A11985) (1:500), GALP Rabbit pAb (A17445) (1:1,000) and GAPDH Rabbit mAb (A19056) (1:2,000) dilution overnight at 4 °C.

Techniques: Transfection, Expressing, Western Blot