Journal: Journal of the American Heart Association

Article Title: Wnt Site Signaling Inhibitor Secreted Frizzled‐Related Protein 3 Protects Mitral Valve Endothelium From Myocardial Infarction–Induced Endothelial‐to‐Mesenchymal Transition

doi: 10.1161/jaha.121.023695

Figure Lengend Snippet: Figure 7. FOXM1 contributes to EndMT and can be suppressed by sFRP3. A, Mitral VECs were treated with sham, post-MI plasma, and post-MI plasma with Siomycin A (1 µmol/L) for 24 hours before immunofluorescence staining using FOXM1 antibody (green; scale bar: 20 µm). B, Percentage of the cells positive for nuclear FOXM1 per total nuclei from 4 independent immunofluorescence assays were graphed. Statistical analysis was conducted using 1-way ANOVA with Tukey’s multiple comparisons test. C, Mitral VECs were treated with MI plasma (n=6) with and without Siomycin A (1 µmol/L) for 24 hours before qPCR analysis. Mean±SD from 3 independent assays were graphed. P values were calculated using the Wilcoxon signed-rank test and were significant (P<0.05) for all tested genes. D, Mitral VECs were treated with MI plasma (n=6) supplemented with sFRP3 (250 ng/mL) for 24 hours before qPCR analysis. Mean±SD from 3 independent assays were graphed. P value was calculated using nonparametric Mann- Whitney test. E, FOXM1 staining in mitral VECs following exposure to post-MI plasma and post-MI plasma supplemented with 250 ng/mL of sFRP3 for 24 hours. DAPI was used to stain nuclei (scale bar: 50 µm). F, Mean±SD of % cells positive for nuclear FOXM1 per total nuclei from 5 independent immunofluorescence assays with 2 individual post-MI plasma were graphed. P values were calculated using Mann-Whitney test. EndMT indicates endothelial-to-mesenchymal transition; FC, fold changes; FOXM1, forkhead box M1; MI, myocardial infarction; NS, not significant; qPCR, quantitative polymerase chain reaction; sFRP3, secreted frizzled-related protein 3; SioA, Siomycin A; and VEC, valve endothelial cell.

Article Snippet: Sections were then incubated with rabbit antihuman FOXM1 polyclonal antibody (Novus Biologicals, Littleton, CO; No. NBP1- 30961) diluted 1:300 or anti- Ki67 (Abcam; No. 15580) diluted 1:50 in 5% normal horse serum (Vector Laboratories, Burlingame, CA; No. S- 2000) for 90 minutes at room temperature, followed by 45 minutes incubation with antirabbit secondary antibody (LSAB Kit; Dako; No. K0675).

Techniques: Clinical Proteomics, Immunofluorescence, Staining, MANN-WHITNEY, Real-time Polymerase Chain Reaction

Journal: Journal of the American Heart Association

Article Title: Wnt Site Signaling Inhibitor Secreted Frizzled‐Related Protein 3 Protects Mitral Valve Endothelium From Myocardial Infarction–Induced Endothelial‐to‐Mesenchymal Transition

doi: 10.1161/jaha.121.023695

Figure Lengend Snippet: Figure 8. Proposed model of EndMT induction in MV by post-MI plasma. sFRP3-deficient post-MI plasma releases the brake on Wnt signaling, increases FOXM1 transcriptional activity, nuclear localization of Slug, which initiates EndMT within days after MI. EndMT indicates endothelial-to-mesenchymal transition; FOXM1, forkhead box M1; MI, myocardial infarction; MV, mitral valve; NS, not significant; sFRP3, secreted frizzled-related protein 3; TGFβ, transforming growth factor beta; VE, vascular endothelial; and Wnt, wingless-related integration site.

Article Snippet: Sections were then incubated with rabbit antihuman FOXM1 polyclonal antibody (Novus Biologicals, Littleton, CO; No. NBP1- 30961) diluted 1:300 or anti- Ki67 (Abcam; No. 15580) diluted 1:50 in 5% normal horse serum (Vector Laboratories, Burlingame, CA; No. S- 2000) for 90 minutes at room temperature, followed by 45 minutes incubation with antirabbit secondary antibody (LSAB Kit; Dako; No. K0675).

Techniques: Clinical Proteomics, Activity Assay

Journal: eLife

Article Title: CPT1A mediates radiation sensitivity in colorectal cancer

doi: 10.7554/eLife.97827

Figure Lengend Snippet: ( A ) The protein level of FOXM1, CPT1A, catalase (CAT), SOD1, SOD2, SOD3 after knockout and overexpression of CPT1A. ( B ) The mRNA level of FOXM1, CAT, SOD1, SOD2, SOD3 after knockout and overexpression of CPT1A. ( C ) Venn diagram showing the potential transcription factor of SOD1, SOD2, and CAT. ( D ) The protein level of FOXM1, CPT1A, CAT, SOD1, SOD2 after overexpression of FOXM1 in HCT116-CPT1AKO cells. ( E ) Schematic diagram summarising our working model, namely, decreased CPT1A promotes the transcription factor activity of FOXM1, increasing the mRNA and protein level of CAT, SOD1, and SOD2, followed by increasing ROS scavenge after irradiation and therefore colorectal cancer (CRC) cells become radioresistance. ***p<0.001, **p<0.01, *p<0.05. Figure 6—source data 1. Original files for western blot analysis displayed in . Figure 6—source data 2. PDF file containing original western blots for .

Article Snippet: Antibody , FOXM1 Rabbit Polyclonal antibody , Proteintech (China) , 13147-1-AP , .

Techniques: Knock-Out, Over Expression, Activity Assay, Irradiation, Western Blot

Journal: eLife

Article Title: CPT1A mediates radiation sensitivity in colorectal cancer

doi: 10.7554/eLife.97827

Figure Lengend Snippet: ( A ) The correlation of FOXM1 with SOD1. ( B ) The correlation of FOXM1 with SOD2.

Article Snippet: Antibody , FOXM1 Rabbit Polyclonal antibody , Proteintech (China) , 13147-1-AP , .

Techniques:

Journal: eLife

Article Title: CPT1A mediates radiation sensitivity in colorectal cancer

doi: 10.7554/eLife.97827

Figure Lengend Snippet: Potential binding site of SOD1, SOD2, catalase (CAT) promoter predicted by hTFtarget and JASPAR.

Article Snippet: Antibody , FOXM1 Rabbit Polyclonal antibody , Proteintech (China) , 13147-1-AP , .

Techniques: Binding Assay, Sequencing

Journal: eLife

Article Title: CPT1A mediates radiation sensitivity in colorectal cancer

doi: 10.7554/eLife.97827

Figure Lengend Snippet:

Article Snippet: Antibody , FOXM1 Rabbit Polyclonal antibody , Proteintech (China) , 13147-1-AP , .

Techniques: Transfection, Construct, SYBR Green Assay, Protein Extraction, Single Cell Gel Electrophoresis, Activity Assay

Journal: eLife

Article Title: CPT1A mediates radiation sensitivity in colorectal cancer

doi: 10.7554/elife.97827

Figure Lengend Snippet: Figure 6. CPT1A increases the transcription and protein of reactive oxygen species (ROS) scavenge-related genes by regulating the transcription factor activity of FOXM1. (A) The protein level of FOXM1, CPT1A, catalase (CAT), SOD1, SOD2, SOD3 after knockout and overexpression of CPT1A. (B) The mRNA level of FOXM1, CAT, SOD1, SOD2, SOD3 after knockout and overexpression of CPT1A. (C) Venn diagram showing the potential transcription factor of SOD1, SOD2, and CAT. (D) The protein level of FOXM1, CPT1A, CAT, SOD1, SOD2 after overexpression of FOXM1 in HCT116-CPT1AKO cells.

Article Snippet: DOI: https://doi.org/10.7554/eLife.97827 14 of 21 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody SOD1 Rabbit Polyclonal antibody Proteintech (China) 10269- 1- AP Antibody SOD2 Rabbit Polyclonal antibody Proteintech (China) 24127- 1- AP Antibody SOD3 Rabbit Polyclonal antibody Proteintech (China) 14316- 1- AP Antibody Catalase Rabbit Polyclonal antibody Proteintech (China) 21260- 1- AP Antibody FOXM1 Rabbit Polyclonal antibody Proteintech (China) 13147- 1- AP Antibody Anti- rabbit IgG, HRP- linked Antibody Cell Signaling Technology (USA) 7074 Chemical compound, drug TRIzol TakaraBio (Japan) 9108 Commercial assay or kit Evo M- MLV RT Mix Kit Accurate Biotechnology (China) AG11728 Commercial assay or kit SYBR Green Premix Pro Taq HS qPCR Kit Accurate Biotechnology (China) AG11701 Commercial assay or kit Protein extraction kit KeyGen BioTech (China) KGP113- SDS Commercial assay or kit Comet assay kit KeyGen BioTech (China) KGA240 Chemical compound, drug PI Beyotime (China) ST511 Chemical compound, drug DAPI Beyotime (China) C1002 Commercial assay or kit ROS detection kit Beyotime (China) S0033S Commercial assay or kit GSH detection kit Solarbio (China) BC1175 Commercial assay or kit GSSG detection kit Solarbio (China) BC1180 Commercial assay or kit SOD enzyme activity kit Solarbio (China) BC0170 Commercial assay or kit CAT enzyme activity kit Solarbio (China) BC0200 Commercial assay or kit POD enzyme activity kit Solarbio (China) BC0090 Continued

Techniques: Activity Assay, Knock-Out, Over Expression

Journal: OncoTargets and therapy

Article Title: Expression and functional characterization of FOXM1 in non-small cell lung cancer

doi: 10.2147/OTT.S162523

Figure Lengend Snippet: The FOXM1 protein was detected in NSCLC tissues by immunohistochemistry (×100). Notes: ( A ) FOXM1-positive expression in adenocarcinoma; ( B ) FOXM1-positive expression in squamous cell carcinoma; ( C ) adenocarcinoma without FOXM1 expression; and ( D ) squamous cell carcinoma without FOXM1 expression. Abbreviation: NSCLC, non-small cell lung carcinoma.

Article Snippet: The extracted total protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane, which was subsequently immersed in 5% skim-milk blocking solution for 2 h. The membrane was then incubated with a rabbit anti-human FOXM1 polyclonal antibody and mouse anti-human GAPDH monoclonal antibody (1:1,000; Santa Cruz Biotechnologies, Dallas, TX, USA) separately at 4°C overnight, before being exposed to a secondary antibody at 25°C for 1 h. Finally, chemiluminescence reagent was added.

Techniques: Immunohistochemistry, Expressing

Journal: OncoTargets and therapy

Article Title: Expression and functional characterization of FOXM1 in non-small cell lung cancer

doi: 10.2147/OTT.S162523

Figure Lengend Snippet: The correlations between FOXM1 expression and clinicopathological features in NSCLC patients

Article Snippet: The extracted total protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane, which was subsequently immersed in 5% skim-milk blocking solution for 2 h. The membrane was then incubated with a rabbit anti-human FOXM1 polyclonal antibody and mouse anti-human GAPDH monoclonal antibody (1:1,000; Santa Cruz Biotechnologies, Dallas, TX, USA) separately at 4°C overnight, before being exposed to a secondary antibody at 25°C for 1 h. Finally, chemiluminescence reagent was added.

Techniques: Expressing

Journal: OncoTargets and therapy

Article Title: Expression and functional characterization of FOXM1 in non-small cell lung cancer

doi: 10.2147/OTT.S162523

Figure Lengend Snippet: Univariate analysis of clinicopathological factors for the overall survival in NSCLC patients

Article Snippet: The extracted total protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane, which was subsequently immersed in 5% skim-milk blocking solution for 2 h. The membrane was then incubated with a rabbit anti-human FOXM1 polyclonal antibody and mouse anti-human GAPDH monoclonal antibody (1:1,000; Santa Cruz Biotechnologies, Dallas, TX, USA) separately at 4°C overnight, before being exposed to a secondary antibody at 25°C for 1 h. Finally, chemiluminescence reagent was added.

Techniques: Expressing

Journal: OncoTargets and therapy

Article Title: Expression and functional characterization of FOXM1 in non-small cell lung cancer

doi: 10.2147/OTT.S162523

Figure Lengend Snippet: Survival analysis of 128 NSCLC patients. Notes: ( A ) Differentiation, ( B ) LN metastasis, ( C ) TNM stage, and ( D ) FOXM1 expression. Abbreviations: NSCLC, non-small cell lung carcinoma; LN, lymph node; TNM, tumor-node-metastasis.

Article Snippet: The extracted total protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane, which was subsequently immersed in 5% skim-milk blocking solution for 2 h. The membrane was then incubated with a rabbit anti-human FOXM1 polyclonal antibody and mouse anti-human GAPDH monoclonal antibody (1:1,000; Santa Cruz Biotechnologies, Dallas, TX, USA) separately at 4°C overnight, before being exposed to a secondary antibody at 25°C for 1 h. Finally, chemiluminescence reagent was added.

Techniques: Expressing

Journal: OncoTargets and therapy

Article Title: Expression and functional characterization of FOXM1 in non-small cell lung cancer

doi: 10.2147/OTT.S162523

Figure Lengend Snippet: Univariate and multivariate analyses of prognostic variables for overall survival

Article Snippet: The extracted total protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane, which was subsequently immersed in 5% skim-milk blocking solution for 2 h. The membrane was then incubated with a rabbit anti-human FOXM1 polyclonal antibody and mouse anti-human GAPDH monoclonal antibody (1:1,000; Santa Cruz Biotechnologies, Dallas, TX, USA) separately at 4°C overnight, before being exposed to a secondary antibody at 25°C for 1 h. Finally, chemiluminescence reagent was added.

Techniques: Expressing

Journal: OncoTargets and therapy

Article Title: Expression and functional characterization of FOXM1 in non-small cell lung cancer

doi: 10.2147/OTT.S162523

Figure Lengend Snippet: The FOXM1 expression in NSCLC cell lines and their biological functions. Notes: ( A ) The FOXM1 expression in NSCLC cell lines and normal bronchial epithelial cell line HBE was detected by Western blot (left) and ELISA (right). Western blot ( B ) and ELISA ( C ) verified the efficiency of FOXM1 overexpression and knockdown in NSCLC cell lines. ( D ) The FOXM1 overexpression enhanced proliferation of NSCLC cell lines. ( E ) The FOXM1 overexpression promoted invasion of NSCLC cell lines. (* P <0.05; the t -test was used to contrast quantitative variables between groups). Abbreviations: NSCLC, non-small-cell lung carcinoma; LN, lymph node; TNM, tumor-node-metastasis.

Article Snippet: The extracted total protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane, which was subsequently immersed in 5% skim-milk blocking solution for 2 h. The membrane was then incubated with a rabbit anti-human FOXM1 polyclonal antibody and mouse anti-human GAPDH monoclonal antibody (1:1,000; Santa Cruz Biotechnologies, Dallas, TX, USA) separately at 4°C overnight, before being exposed to a secondary antibody at 25°C for 1 h. Finally, chemiluminescence reagent was added.

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Over Expression