Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

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: A , Hierarchical clustering and heat map of normalized read counts of 206 significantly regulated mRNA transcripts in mitral VECs treated with naïve (n=3) and post‐MI plasma (n=3). Red color represents higher expression and green color indicates the lower expression. One minus Pearson correlation metric was used for hierarchical clustering. B , Hierarchical clustering and heat map of log2 fold change of FOXM1‐related genes. Red color represents the higher expression and green color indicates the lower expression for each mRNA. One minus Pearson correlation metric was used for hierarchical clustering. C , Ovine mitral VECs were exposed to naïve (n=3), sham (n=3), and MI (n=6) plasma for 24 hours before qPCR analysis. All values were normalized to average naïve and graphed. Data represent mean±SD of 3 independent assays. P values were calculated using nonparametric Mann‐Whitney test. D , Equal numbers of ovine mitral VECs were seeded and treated with sham (n=4) or MI (n=5) plasma for 24 hours before cell count. Fold change was calculated using average number of cells in triplicates for all sham controls. Mean±SD from 2 independent assays were graphed. P values were calculated using nonparametric Mann‐Whitney test. E through G , Mitral valve tissue from animals with naïve ( E ), sham ( F ), and inferior MI ( G ) were stained for FOXM1 expression using immunohistochemistry (scale bar: 500 µm). H , Number of cells expressing FOXM1 divided by total nuclei from 10 fields per each individual section was graphed. P values were calculated using 1‐way ANOVA with Tukey’s multiple comparisons test. I , Post‐MI MV was costained with anti‐FOXM1 (red) and anti‐CD31 (green) using immunofluorescence staining and DAPI staining to visualize nuclei. Arrows indicate FOXM1+ nuclei. scale bar: 50 µm. α‐SMA indicates α‐smooth muscle actin; CCNB1, Cyclin B1; FC, fold changes; FOXM1, forkhead box M1; MI, myocardial infarction; MV, mitral valve; qPCR, quantitative polymerase chain reaction; sFRP3, secreted frizzled‐related protein 3; TGFβ, transforming growth factor beta; VE, vascular endothelial; 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, Expressing, MANN-WHITNEY, Cell Counting, Staining, Immunohistochemistry, Immunofluorescence, Real-time Polymerase Chain Reaction

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

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: 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: Cardiovascular and Cerebrovascular Disease

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: 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: Scientific Reports

Article Title: FOXM1: a new therapeutic target of extramammary Paget disease

doi: 10.1038/s41598-024-54773-8

Figure Lengend Snippet: FOXM1 expression in patients’ EMPD tissues. ( A ) Immunohistochemical images of FOXM1 in patients’ EMPD tissues. Representative images of early lesions [tumor thickness (TT) ≤ 3 mm, n = 102], locally advanced lesions (TT > 3 mm, n = 10), and metastatic lesions (n = 17) are shown. Scale bars = 100 μm. ( B ) Comparison of FOXM1-positive cells (%) among early, locally advanced, and metastatic lesions of patients’ tissues. * p < 0.05 and *** p < 0.001. ( C ) Survival curves of patients with ≤ 10% (n = 77) or > 10% (n = 35) FOXM1 staining positivity in immunohistochemistry. Patients with FOXM1 > 10% had significantly shorter disease-specific survival than those with FOXM1 ≤ 10% ( p = 0.0397).

Article Snippet: Antibodies used were rabbit anti-human FOXM1 (1:1000, 5436; Cell Signaling Technology), rabbit anti-human cyclin B1 (1:1000, 12231, Cell Signaling Technology), and rabbit anti-human β-actin (1:2000, 4970; Cell Signaling Technology).

Techniques: Expressing, Immunohistochemical staining, Comparison, Staining, Immunohistochemistry

Journal: Scientific Reports

Article Title: FOXM1: a new therapeutic target of extramammary Paget disease

doi: 10.1038/s41598-024-54773-8

Figure Lengend Snippet: FOXM1 is highly expressed in KS-EMPD-1 cells. ( A ) FOXM1 mRNA expression in KS-EMPD-1 cells and NHEKs. Mean ± SD of FOXM1 obtained from three independent experiments is shown. *** p < 0.001. ( B ) FOXM1 protein expression in KS-EMPD-1 cells and NHEKs. Representative blot images (left) and mean ± SD of FOXM1 protein (right) are shown. Experiments were independently repeated three times. Original, uncropped images are shown in Supplementary Fig. . *** p < 0.001. ( C ) Immunocytochemical images of FOXM1 in KS-EMPD-1 cells and NHEKs. FOXM1 (green), DAPI (blue, nuclear), and merged images of FOXM1 and DAPI are shown. Scale bars = 100 μm.

Article Snippet: Antibodies used were rabbit anti-human FOXM1 (1:1000, 5436; Cell Signaling Technology), rabbit anti-human cyclin B1 (1:1000, 12231, Cell Signaling Technology), and rabbit anti-human β-actin (1:2000, 4970; Cell Signaling Technology).

Techniques: Expressing

Journal: Scientific Reports

Article Title: FOXM1: a new therapeutic target of extramammary Paget disease

doi: 10.1038/s41598-024-54773-8

Figure Lengend Snippet: KS-EMPD-1 is sensitive to a FOXM1 inhibitor, thiostrepton. KS-EMPD-1 cells and NHEKs were incubated with DMSO (0.1%) or various concentrations of thiostrepton (0.25–10.0 μM) for 72 h and evaluated for the number of viable cells using a formazan-based method. Mean ± SD of fold change (viable cells) calculated from three independent experiments is shown. The IC 50 of thiostrepton is shown in the boxes below the graphs. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Antibodies used were rabbit anti-human FOXM1 (1:1000, 5436; Cell Signaling Technology), rabbit anti-human cyclin B1 (1:1000, 12231, Cell Signaling Technology), and rabbit anti-human β-actin (1:2000, 4970; Cell Signaling Technology).

Techniques: Incubation

Journal: Scientific Reports

Article Title: FOXM1: a new therapeutic target of extramammary Paget disease

doi: 10.1038/s41598-024-54773-8

Figure Lengend Snippet: FOXM1 knockdown inhibits proliferation, migration, and invasion of KS-EMPD-1 cells. Cells were transfected with FOXM1 siRNA and evaluated for their proliferation, migration, and invasion. ( A ) Knockdown efficiency of FOXM1 mRNA. Mean ± SD of FOXM1 calculated from three independent experiments is shown. *** p < 0.001. ( B ) Knockdown efficiency of FOXM1 protein. Representative blot images (upper) and mean ± SD of FOXM1 protein (lower) are shown. Experiments were independently repeated three times. Original, uncropped images are shown in Supplementary Fig. . * p < 0.05, ** p < 0.01, and *** p < 0.001. ( C ) Viable cells in control and FOXM1 siRNA-transfected conditions were quantified using a formazan-based method. Experiments were independently repeated three times. *** p < 0.001. ( D ) CCNB1 mRNA expression in control or FOXM1 siRNA-transfected cells at 24 h post-transfection. Mean ± SD of FOXM1 expression calculated from three independent experiments is shown. *** p < 0.001. ( E ) Cyclin B1 protein expression in control or FOXM1 siRNA-transfected cells at 48 h post-transfection. Experiments were independently repeated three times and representative blot images (upper) and mean ± SD of cyclin B1 protein expression (lower) are shown. Original, uncropped images are shown in Supplementary Fig. . * p < 0.05. Cells were transfected with siRNA for 48 h. The cell monolayers were then scratched and the wound area were captured for 24 h. Representative images ( F ) and wound area relative to that at 0 h ( G ) calculated from three independent experiments are shown. * p < 0.05 and ** p < 0.01. Cells were transfected with siRNA for 48 h and evaluated for cell invasion. Representative images ( H ) and mean ± SD of absorbance of cell staining dye ( I ) are shown. Experiments were independently repeated three times. Scale bars = 1.0 mm. *** p < 0.001.

Article Snippet: Antibodies used were rabbit anti-human FOXM1 (1:1000, 5436; Cell Signaling Technology), rabbit anti-human cyclin B1 (1:1000, 12231, Cell Signaling Technology), and rabbit anti-human β-actin (1:2000, 4970; Cell Signaling Technology).

Techniques: Knockdown, Migration, Transfection, Control, Expressing, Staining

Journal: Scientific Reports

Article Title: FOXM1: a new therapeutic target of extramammary Paget disease

doi: 10.1038/s41598-024-54773-8

Figure Lengend Snippet: Knockdown of FOXM1 increases the chemosensitivity of KS-EMPD-1 cells. KS-EMPD-1 cells were transfected with control or FOXM1 siRNA for 24 h and further treated with vehicle control or anticancer drugs for 72 h. The viable cells were then quantitated using a formazan-based method. 5-Fluorouracil (5-FU, final concentration 100 nM), docetaxel (DTX, final concentration 5 μM), and paclitaxel (PTX, final concentration 1 μM) were dissolved in DMSO and cisplatin (CDDP, final concentration 10 μM) was dissolved in saline and further added to the culture medium. Concentration of anticancer drugs was determined based on the C max of each drug. Mean ± SD of fold change of viable cells calculated from three independent experiments is shown. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Antibodies used were rabbit anti-human FOXM1 (1:1000, 5436; Cell Signaling Technology), rabbit anti-human cyclin B1 (1:1000, 12231, Cell Signaling Technology), and rabbit anti-human β-actin (1:2000, 4970; Cell Signaling Technology).

Techniques: Knockdown, Transfection, Control, Concentration Assay, Saline

Journal: The Journal of pathology

Article Title: MiR-506 Suppresses Proliferation and Induces Senescence by Directly Targeting the CDK4/6-FOXM1 Axis in Ovarian Cancer

doi: 10.1002/path.4348

Figure Lengend Snippet: A. Representative images of immunohistochemical staining for Ki-67, CDK4, CDK6, and FOXM1 in cases expressing low or high levels of miR-506 are shown. Scale bar represents 100μm.

Article Snippet: Rabbit anti-human monoclonal antibody against Ki-67 (1:1500, ab15580; Abcam, Cambridge, MA), rabbit anti-human polyclonal antibody against CDK4 (1:250, c-22; Santa Cruz Biotechnology), rabbit anti-human polyclonal antibody against CDK6 (1:100, ab-13; Sigma), rabbit anti-human polyclonal antibody against FOXM1 (1:400, c-20; Santa Cruz Biotechnology), and EnVision+ System-HRP rabbit (Dako, Carpinteria, CA) were used.

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: The Journal of pathology

Article Title: MiR-506 Suppresses Proliferation and Induces Senescence by Directly Targeting the CDK4/6-FOXM1 Axis in Ovarian Cancer

doi: 10.1002/path.4348

Figure Lengend Snippet: Results from this study and the literature support a central role for miR-506 in two cross-talking processes important for cancer. miR-506 directly targets the CDK4/6-FOXM1 axis in promoting cellular senescence, as represented by SA-β-Gal positivity (blue cells). miR-506 inhibits EMT by directly targeting SNAI2, resulting in augmented expression of E-cadherin protein as represented by the cobblestone morphology of epithelial cells (green color) and blockage of invasive mesenchymal cells (red color). Decreased expression of miR-506 can be a result of promoter methylation[12].

Article Snippet: Rabbit anti-human monoclonal antibody against Ki-67 (1:1500, ab15580; Abcam, Cambridge, MA), rabbit anti-human polyclonal antibody against CDK4 (1:250, c-22; Santa Cruz Biotechnology), rabbit anti-human polyclonal antibody against CDK6 (1:100, ab-13; Sigma), rabbit anti-human polyclonal antibody against FOXM1 (1:400, c-20; Santa Cruz Biotechnology), and EnVision+ System-HRP rabbit (Dako, Carpinteria, CA) were used.

Techniques: Expressing, Methylation

Journal: Molecular therapy. Nucleic acids

Article Title: Enhanced HDR-mediated correction of heterozygous COL7A1 mutations for recessive dystrophic epidermolysis bullosa.

doi: 10.1016/j.omtn.2025.102472

Figure Lengend Snippet: Figure 4. COL7A1 transcription and C7 protein analysis in HDR-edited RDEB cells (A) COL7A1 transcript analysis: ONT-seq data of COL7A1 transcripts aligned to the reference genome, displayed as coverage tracks in IGV. Rows from top to bottom: Transcripts from RDEB03-Ks edited with either high-SNP or low-SNP HDR templates, unedited RDEB03-Ks (UN), and normal human keratinocytes (NHKs). The target 11bp deletion (c.8698_8708del) is indicated with a black asterisk, the green asterisk indicates the non-target mutation (c.8780G>A) on the opposite allele. An aberrant splice site is marked by a red arrow. (B) Immunocytochemistry analysis of C7 (in red) in unedited and edited RDEB03-Ks and NHKs as labeled. RDEB03-Ks were edited with low-SNP template with M3814. Nuclei are stained with DAPI (blue). Nuclear FOXM1 staining (green) marks potential progenitor cells. Merged images are shown on the right-hand panel, where cyan represents merged DAPI (blue) and FOXM1 (green) nuclear stains. Scale bar (bottom right), 100 mM. On the far right, images are enlarged with white arrows indicating cells co-stained for FOXM1 and C7. (C) Flow cytometry analysis of C7 expression in HDR-edited RDEB03-Ks. Plots show C7 expression on the x axis and side scatter (SSC-A) on the y axis. The percentage of C7-positive cells is indicated in red text. (Top row) NHKs, unedited RDEB03-Ks (UN), and high-SNP edited RDEB03-Ks without M3814. (Bottom row) RDEB03-Ks edited with low-SNP, med-SNP, and high-SNP HDR templates with M3814. (D) Linear correlation (R2 = 0.9782) between HDR efficiency as assessed by ONT-seq/CRISPResso2 (x axis) and the percentage of C7-positive cells as assessed by flow cytometry (y axis). The y intercept set to 0. (E) The average mean fluorescence intensity (MFI) of the intracellular C7 signal above the baseline of unedited RDEB03 cells in NHKs (n = 3), RDEB03-Ks edited with the Low-SNP (n = 3), Med-SNP (n = 1), and High-SNP templates (n = 3).

Article Snippet: Cells were then blocked with 0.25% Casein in TBS containing 10% human serum (Thermo Fisher Scientific) for 30 min. For the primary antibody co-stains, a human-specific mouse IgG1 anti-C7 antibody (LH7.2, Invitrogen) at a 1:200 dilution and a rabbit anti-FOXM1 antibody (D3F2B, Cell Signaling, NEB) at a 1:200 dilution were prepared in TBS with 10% human serum.

Techniques: Mutagenesis, Immunocytochemistry, Labeling, Staining, Flow Cytometry, Expressing, Cytometry

Journal: CNS Neuroscience & Therapeutics

Article Title: Curcumin Suppresses Malignant Glioma Cells Growth and Induces Apoptosis by Inhibition of SHH / GLI 1 Signaling Pathway in Vitro and Vivo

doi: 10.1111/cns.12163

Figure Lengend Snippet: Curcumin inhibited SHH/GLI1 signaling and its downstream targets oncoproteins in time‐ and dose‐dependent manner. (A) Real‐time everse transcription‐PCR analysis of total RNA from T98G cells following 40 uM of curcumin treatment for different time points (0, 8, 16, 24 h) revealed decreasing expression of Shh, Smo, and GLI1 on mRNA level. (B) Western blot assay showed curcumin treatment led to decreased levels of Shh, GLI1, and GLI1‐regulated target (CyclinD1, Bcl‐2, Foxm1) proteins in time‐dependent manner in T98G cells following 40 uM agent exposure for 24 h. (C) U87 and T98G cells were plated and grown for 24 h and then treated with the solvent DMSO (control) or with different (indicated) doses of curcumin dissolved in DMSO for 24 h before harvesting. Western blot assay shows curcumin inhibits core components of the SHH/GLI1 signaling (SHH, GLI1) and its target oncoproteins (CyclinD1, Bcl‐2, Foxm1) in dose‐dependent manner. Data are shown as mean ± SEM for the three replicates. Statistical significance levels are indicated as: *P < 0.05; **P < 0.01 and ***P < 0.001.

Article Snippet: The membrane was blocked in 5% milk–TBST solution and incubated separately with rabbit antihuman Shh (1:500, Bioss Inc, USA), rabbit antihuman GLI1 (1:1000, Cell Signaling Technology, USA), mouse antihuman CyclinD1 antibody (1:300, Santa Cruz, USA),rabbit antihuman Bcl‐2(1:500, Santa Cruz, USA), rabbit antihuman Foxm1(1:1000, Cell Signaling Technology, USA), rabbit antihuman Bax (1:500, Santa Cruz, USA), mouse antihuman pro‐caspase‐9 (1:500, Santa Cruz, USA), rabbit antihuman pro‐caspase‐3 (1:500, Santa Cruz, USA), or mouse antihuman GAPDH antibody (1:1000, Sigma, USA).

Techniques: Expressing, Western Blot