Journal: FEBS letters

Article Title: miR-134 inhibits epithelial to mesenchymal transition by targeting FOXM1 in non-small cell lung cancer cells.

doi: 10.1016/j.febslet.2012.09.016

Figure Lengend Snippet: Fig. 3. miR-134 directly targets FOXM1 by binding to its 30UTR. (A) The predicted miR-134 binding site within FOXM1 30UTR and its mutated version by site mutagenesis are as shown. (B) Variable FOXM1 expression in four NSCLC cell lines was obtained by Western blot. (C) In comparison with negative controls, miR-134 inhibited FOXM1 protein expression, while reduction of miR-134 by inhibitors moderately restored FOXM1 expression. (D) Luciferase assays indicated that miR-134 downregulated the expression of FOXM1 by binding with its 30UTR.

Article Snippet: Antibodies against FOXM1 (Novus Biologicals, CO., USA), b-actin (Beijing Zhongshan Biotechnology, Beijing, China), E-cadherin and vimentin (Santa Cruz, CA) were used in western analysis in accordance with the manufacturer’s instruction.

Techniques: Binding Assay, Mutagenesis, Expressing, Western Blot, Comparison, Luciferase

Journal: FEBS letters

Article Title: miR-134 inhibits epithelial to mesenchymal transition by targeting FOXM1 in non-small cell lung cancer cells.

doi: 10.1016/j.febslet.2012.09.016

Figure Lengend Snippet: Fig. 4. FOXM1 is involved in TGF-b1-induced EMT in A549 cells. A549 cells were transfected with NC, si-FOXM1 or miR-134 respectively, after 24 h, TGF-b1 (5 ng/ml) was added, and cells were further incubated for 48 h. (A) Western blot analysis of epithelial marker E-cadherin and mesenchymal marker vimentin. At the same time, A549 cells morphological changes were examined. The effect of TGF-b1 on FOXM1 (B) and miR-134 expression (C) were examined. (D) The inhibition effect of miR-134 on EMT was rescued when FOXM1 expression vector was cotransfected.

Article Snippet: Antibodies against FOXM1 (Novus Biologicals, CO., USA), b-actin (Beijing Zhongshan Biotechnology, Beijing, China), E-cadherin and vimentin (Santa Cruz, CA) were used in western analysis in accordance with the manufacturer’s instruction.

Techniques: Transfection, Incubation, Western Blot, Marker, Expressing, Inhibition, Plasmid Preparation

Journal: Cell Reports Medicine

Article Title: L858R emerges as a potential biomarker predicting response of lung cancer models to anti-EGFR antibodies: Comparison of osimertinib vs. cetuximab

doi: 10.1016/j.xcrm.2023.101142

Figure Lengend Snippet: Unlike cells expressing other EGFR mutants, the FOXM1 pathway and compensatory RTKs are inhibited in L858R expressors following cetuximab treatment (A–C) H3255 (L858R-EGFR), 11-18 (L858R-EGFR), and PC9 cells (Del19-EGFR) were treated for 24 h with cetuximab (10 μg/mL), or erlotinib or osimertinib (500 nM for 11-18; 50 nM for the other cell lines). Protein extracts were resolved and probed with antibodies specific to the indicated receptors and downstream pathways. GAPDH was used as the loading control. (D–F) The indicated cell lines, including PC9ER (Del19 and T790M EGFR), were treated for 48 h with cetuximab (10 μg/mL), erlotinib, or osimertinib (each at 50 nM). Protein extracts were resolved, blotted, and probed with antibodies specific to components of the FOXM1 and other pathways. GAPDH and vinculin were used to ensure equal gel loading.

Article Snippet: FOXM1, Rabbit monoclonal , Cell Signaling Technology , Cat#5436; RRID: AB_10692483.

Techniques: Expressing, Control

Journal: Cell Reports Medicine

Article Title: L858R emerges as a potential biomarker predicting response of lung cancer models to anti-EGFR antibodies: Comparison of osimertinib vs. cetuximab

doi: 10.1016/j.xcrm.2023.101142

Figure Lengend Snippet:

Article Snippet: FOXM1, Rabbit monoclonal , Cell Signaling Technology , Cat#5436; RRID: AB_10692483.

Techniques: Derivative Assay, Recombinant, Generated, Transfection, Plasmid Preparation, Software

Journal: Molecular Therapy. Nucleic Acids

Article Title: miR-199a-3p increases the anti-tumor activity of palbociclib in liver cancer models

doi: 10.1016/j.omtn.2022.07.015

Figure Lengend Snippet: miR-199a-3p increased palbociclib efficacy on human HCC cells in vitro and in vivo (A) Hep3B cells were transduced with an Adeno Associated Virus expressing miR-199a-3p (AAVV-199) or with a control AAVV (AAVV-CTRL) at MOI = 100. A group of cells received no treatment (NT). Palbociclib (PB) (20 μM) was added to cell culture 72 h before cell collection. The levels of miR-199a-3p were measured 120 h after transduction. (B) Viability and apoptosis were evaluated 120 h after transduction. Data are represented as mean +SD. (C) Western blot analysis and quantification of RB1, AKT, FOXM1, and their phosphorylated forms in Hep3B-treated cells. The values were normalized on the GAPDH expression. Because Hep3B cells exhibit a low level of full-length protein, digital images of RB1 and p-RB1 were acquired with an exposure time of 300 s instead of 30 s. (D) Tumor nodule volumes in DEN-treated TG221 mice of the following groups: (1) palbociclib (PB) (n = 11); (2) miR-199a-3p mimics (miR-199) (n = 9); (3) palbociclib + miR-199a-3p mimics (PB + miR-199) (n = 11); (4) sorafenib (SF) (n = 9); (5) vehicle/scramble oligonucleotides (CTRL) (n = 9). Experimental therapies started at 6 months, when all mice presented one or more tumor nodules in their livers. Single tumor nodules were monitored by ultrasound at the beginning and end of treatment. (E) Distribution of tumor nodule size measured at the time of euthanization. (F) Mice weight was monitored during treatment to check drug toxicity. Data are represented as mean ± SD. ∗p value ≤0.05; ∗∗p value ≤0.01; ∗∗∗p value ≤0.001; ∗∗∗∗p value ≤0.0001.

Article Snippet: After incubation with 5% Blocking agent, the membrane were incubated overnight at 4°C with the following antibodies and specific conditions: Rabbit antibodies against p-RB (Ser780, D59B7, #8180), p-AKT (Ser473, D9E XP, #4060), p-FOXM1 (Thr600, D9M6G, #14655), RB (D20, #9313), FOXM1 (D12D5, #5436) and PAK4 (#3242) were all from Cell Signaling Technology (Danvers, MA, USA) and were diluted in 5% w/v BSA (A4503, Sigma-Aldrich), 1X Tris-buffered saline (TBS; Bio-Rad Laboratories, Hercules, CA, USA), and 0.1% Tween 20 (Bio-Rad) and incubated at 4°C for 16 h. Rabbit antibody against AKT (C-20, sc-1618) was obtained from Santa Cruz Biotechnology (Dallas, TX, USA) and was diluted in 1% w/v milk, 1X TBS, and 0.1% Tween 20 (Bio-Rad).

Techniques: In Vitro, In Vivo, Transduction, Virus, Expressing, Control, Cell Culture, Western Blot

Journal: Cancer Research

Article Title: Down-regulation of Forkhead Box M1 Transcription Factor Leads to the Inhibition of Invasion and Angiogenesis of Pancreatic Cancer Cells

doi: 10.1158/0008-5472.can-07-1265

Figure Lengend Snippet: Figure 1. FoxM1 expression in PC cell lines. CS, control siRNA; FS, FoxM1 siRNA; CP, control plasmid; FP, FoxM1 cDNA plasmid. A and B, FoxM1 mRNA and protein levels were measured by real-time RT-PCR and Western blotting, respectively. C, FoxM1 mRNA levels were down-regulated by FoxM1 siRNA. Columns, mean of three independent experiments; bars, SD. D, FoxM1 protein levels were down-regulated by siRNA and up-regulated by FoxM1 cDNA plasmid in three PC cells, respectively. *, P < 0.05 relative to control.

Article Snippet: FoxM1 siRNA and siRNA control were obtained from Santa Cruz Biotechnology.

Techniques: Expressing, Control, Plasmid Preparation, Quantitative RT-PCR, Western Blot

Journal: Cancer Research

Article Title: Down-regulation of Forkhead Box M1 Transcription Factor Leads to the Inhibition of Invasion and Angiogenesis of Pancreatic Cancer Cells

doi: 10.1158/0008-5472.can-07-1265

Figure Lengend Snippet: Figure 2. Effects of altered FoxM1 expression on PC cell growth. Top, inhibition of cancer cell growth by FoxM1 siRNA tested by MTT assay. Bottom, promotion of cancer cell growth by FoxM1 cDNA tested by MTT assay. Points, means of three separate experiments having six determinations per experiment for each experimental condition; bars, SD. *, P < 0.05, relative to control.

Article Snippet: FoxM1 siRNA and siRNA control were obtained from Santa Cruz Biotechnology.

Techniques: Expressing, Inhibition, MTT Assay, Control

Journal: Cancer Research

Article Title: Down-regulation of Forkhead Box M1 Transcription Factor Leads to the Inhibition of Invasion and Angiogenesis of Pancreatic Cancer Cells

doi: 10.1158/0008-5472.can-07-1265

Figure Lengend Snippet: Figure 5. A and B, real-time RT-PCR and Western blot analysis showed that FoxM1 siRNA inhibited the expression of MMP-2, MMP-9, and uPAR genes at mRNA and protein levels in PC cells. C, FoxM1 siRNA inhibited the activity of MMP-2 in PC cells. D, FoxM1 siRNA inhibited the activity of MMP-9 in BxPC-3 PC cells. E, FoxM1 siRNA inhibited the activity of VEGF in PC cells. The experiments were repeated thrice. *, P < 0.05; **, P < 0.01, relative to control.

Article Snippet: FoxM1 siRNA and siRNA control were obtained from Santa Cruz Biotechnology.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Activity Assay, Control

Journal: Cancer Research

Article Title: Down-regulation of Forkhead Box M1 Transcription Factor Leads to the Inhibition of Invasion and Angiogenesis of Pancreatic Cancer Cells

doi: 10.1158/0008-5472.can-07-1265

Figure Lengend Snippet: Figure 6. FoxM1 siRNA decreased PC cell migration and invasion and reduced the HUVECs tube formation. The experiments were repeated thrice. *, P < 0.05 relative to control. A, top, migration assay showing that FoxM1 siRNA decreased PC cell migration. Bottom, value of fluorescence from the migrated cells. B, top, invasion assay showing that FoxM1 siRNA-transfected cells resulted in low penetration through the Matrigel-coated membrane, compared with control cells. Bottom, value of fluorescence from the invaded cells. The value indicated the comparative amount of invaded cells. C, top, Conditioned media from FoxM1 siRNA-transfected BxPC-3 and HPAC cells were able to significantly reduce the tube formation of HUVECs in 6 h incubation compared with the medium from control. Bottom, image analysis of tubule/capillary length was carried out using software image analysis program Scion Image. Quantification of cumulative tube length of endothelial cells.

Article Snippet: FoxM1 siRNA and siRNA control were obtained from Santa Cruz Biotechnology.

Techniques: Migration, Control, Fluorescence, Invasion Assay, Transfection, Membrane, Incubation, Software

Journal: Oncology reports

Article Title: Apoptosis induced by 7-difluoromethoxyl-5,4'-di-n-octyl genistein via the inactivation of FoxM1 in ovarian cancer cells.

doi: 10.3892/or.2012.1739

Figure Lengend Snippet: Figure 3. The downregulation of FoxM1 expression by DFOG and genistein at mRNA level using RT-PCR in SKOV3 (A) and CoC1 (C) cell lines and protein level using western blotting in SKOV3 (B) and CoC1 (D) cell lines is shown.

Article Snippet: Primary antibodies for FoxM1, survivin, cyclin B, p27KIP1, CDC25B, and β-actin, as well as horseradish peroxidase-conjugated rabbit anti-mouse secondary antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Oncology reports

Article Title: Apoptosis induced by 7-difluoromethoxyl-5,4'-di-n-octyl genistein via the inactivation of FoxM1 in ovarian cancer cells.

doi: 10.3892/or.2012.1739

Figure Lengend Snippet: Figure 4. Shown is the modulation of the protein expressions of FoxM1 downstream target genes by DFOG and genistein in SKOV3 (A) and CoC1 (B) cell lines.

Article Snippet: Primary antibodies for FoxM1, survivin, cyclin B, p27KIP1, CDC25B, and β-actin, as well as horseradish peroxidase-conjugated rabbit anti-mouse secondary antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques:

Journal: Oncology reports

Article Title: Apoptosis induced by 7-difluoromethoxyl-5,4'-di-n-octyl genistein via the inactivation of FoxM1 in ovarian cancer cells.

doi: 10.3892/or.2012.1739

Figure Lengend Snippet: Figure 6. FoxM1 cDNA transfection reduces the downregulation of FoxM1 protein expression by DFOG and genistein using western blot analysis (A), inhibi tion of cell growth by MTT assay (B), induction of histone/DNA fragment levels using ELISA (C), and increase of Sub-G1 population using flow cytometry analysis (D) in SKOV3 cell line. *P<0.05 vs. treatment with DMSO; #P<.05 vs. treatment with 5 µmol/l DFOG or FoxM1 cDNA transfection alone.

Article Snippet: Primary antibodies for FoxM1, survivin, cyclin B, p27KIP1, CDC25B, and β-actin, as well as horseradish peroxidase-conjugated rabbit anti-mouse secondary antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Transfection, Expressing, Western Blot, MTT Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Oncology reports

Article Title: Apoptosis induced by 7-difluoromethoxyl-5,4'-di-n-octyl genistein via the inactivation of FoxM1 in ovarian cancer cells.

doi: 10.3892/or.2012.1739

Figure Lengend Snippet: Figure 5. FoxM1 siRNA enhances the downregulation of FoxM1 protein expression by DFOG and genistein using western blot analysis (A), inhibition of cell growth by MTT assay (B), induction of histone/DNA fragment levels using ELISA (C), and increase of the Sub-G1 population using flow cytometry analysis (D) in SKOV3 cell line. *P<0.05 vs. treatment with DMSO; *P<0.05 vs. treatment with 5 µmol/l DFOG or FoxM1 siRNA alone.

Article Snippet: Primary antibodies for FoxM1, survivin, cyclin B, p27KIP1, CDC25B, and β-actin, as well as horseradish peroxidase-conjugated rabbit anti-mouse secondary antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Western Blot, Inhibition, MTT Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Cancer Medicine

Article Title: The Preclinical Effects and Mechanisms of Biofield Therapy on Pancreatic Cancer Cell Growth and Metastasis

doi: 10.1002/cam4.71726

Figure Lengend Snippet: Expression of proteins that regulate the cell cycle and cell signaling in PANC‐1 cells exposed to biofield therapy (BT) for 15 min, compared with sham control (SC) and incubator control (IC). (A) Heatmap of cell cycle regulating proteins and cell signaling proteins in PANC‐1 cells, analyzed by reverse phase protein array (RPPA). The proteins included in this heatmap were statistically significant among different treatment groups according to Wilcoxon rank‐sum analysis, with p values lower than the Benjamini‐Hochberg thresholds for false discovery rate (< 0.05). (B) Expression of cell cycle regulating proteins and epigenetic modification proteins, such as CDK1, cyclin B1, FOXM1, and ALKBH5 in PANC‐1 cells exposed to BT compared with SC, as examined by RPPA ( n = 7). (C‐D) Western blot analysis of FOXM1 (C) and p21 (D) in PANC‐1 cells exposed to BT for 15 min. Data are presented as mean ± SD (*** p < 0.001; **** p < 0.0001; n = 7).

Article Snippet: The transfection of a scramble control vector or a FOXM1 knockout gene kit via CRISPR‐Cas9 (OriGene, cat. # KN402246 ) was carried out using TurboFectin Transfection Reagent (OriGene, cat. #TF81001) following the manufacturer's instructions.

Techniques: Expressing, Control, Protein Array, Modification, Western Blot

Journal: Cancer Medicine

Article Title: The Preclinical Effects and Mechanisms of Biofield Therapy on Pancreatic Cancer Cell Growth and Metastasis

doi: 10.1002/cam4.71726

Figure Lengend Snippet: Modification of FOXM1 in biofield therapy (BT) elicited cell cycle alteration and anti‐invasiveness in PDAC cells. (A) Western blot analysis of FOXM1 in PANC‐1 cells (WT), FOXM1 stable‐knockdown PANC‐1 cells (KD), or FOXM1‐overexpressing (OE) PANC‐1 cells. (B) Cell cycle of FOXM1 knockout (KO) PANC‐1 cells exposed to BT compared with parental PANC‐1 cells. (C) Invasiveness of parental PANC‐1 cells and FOXM1‐knockdown (KD) PANC‐1 cells exposed to BT. (D) Invasiveness of wild‐type (WT) L3.7 cells and FOXM1‐knockout (KO) L3.7 cells exposed to BT. (E) Invasiveness of WT PANC‐1 cells and FOXM1‐KO PANC‐1 cells exposed to BT. (F) Invasiveness of control COLO357 cells and FOXM1 stably overexpressing COLO357 cells exposed to BT. (G) Invasiveness of control COLO357 and Tet‐inducible FOXM1 overexpressing COLO357 cells exposed to BT. Data are presented as mean ± SD (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).

Article Snippet: The transfection of a scramble control vector or a FOXM1 knockout gene kit via CRISPR‐Cas9 (OriGene, cat. # KN402246 ) was carried out using TurboFectin Transfection Reagent (OriGene, cat. #TF81001) following the manufacturer's instructions.

Techniques: Modification, Western Blot, Knockdown, Knock-Out, Control, Stable Transfection

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: FOXM1 Protects Against Myocardial Ischemia-Reperfusion Injury in Rodent and Porcine Models by Suppressing MKRN1-Dependent LKB1 Ubiquitination.

doi: 10.1002/advs.202508673

Figure Lengend Snippet: Figure 1. Foxm1 expression was elevated in ischemic heart tissues from humans, mice, and pigs. A) Volcano plot showing the gene expression of significantly altered FOX family members based on RNA-seq data from heart samples of WT mice 24 h post-I/R or Sham surgery. B,C) Quantitative real-time polymerase chain reaction (qRT-PCR) analyses of Foxm1 (B) and Foxc2 (C) mRNA levels in heart samples from WT mice 24 h post-I/R or Sham surgery (n = 10). D) Quantitative real-time polymerase chain reaction (qRT-PCR) analyses of Foxm1 mRNA levels in heart samples from normal and ICM patients (n = 7). E) Western blot and quantification of FOXM1 protein levels in heart samples from normal and ICM patients (n = 7). F) Quantitative real-time polymerase chain reaction (qRT-PCR) analyses of Foxm1 mRNA levels in heart samples from Bama pigs 2 months post-I/R or Sham surgery (n = 6 for Sham and n = 8 for I/R). G) Western blot and quantification of FOXM1 protein levels in heart samples from Bama pigs 2 months post-I/R or Sham surgery (n = 6 for Sham and n = 8 for I/R). For all statistical plots, the data are presented as mean ± SD. (B,C) and (E–G) by two-tailed unpaired Student’s t-test. (D) By Welch’s t-test.

Article Snippet: Foxm1 conditional knockin mouse model at the locus of ROSA26 was created by Cyagen Biosciences Inc (Guangzhou, Guangdong, China) using CRISPR/Cas-mediated genome engineering.

Techniques: Expressing, Gene Expression, RNA Sequencing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Two Tailed Test

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: FOXM1 Protects Against Myocardial Ischemia-Reperfusion Injury in Rodent and Porcine Models by Suppressing MKRN1-Dependent LKB1 Ubiquitination.

doi: 10.1002/advs.202508673

Figure Lengend Snippet: Figure 2. Cardiomyocytes-specific FOXM1 deletion results in spontaneous dilated cardiomyopathy. A) Heart sections from Myh6+ and Foxm1fl/flMyh6+

Article Snippet: Foxm1 conditional knockin mouse model at the locus of ROSA26 was created by Cyagen Biosciences Inc (Guangzhou, Guangdong, China) using CRISPR/Cas-mediated genome engineering.

Techniques:

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: FOXM1 Protects Against Myocardial Ischemia-Reperfusion Injury in Rodent and Porcine Models by Suppressing MKRN1-Dependent LKB1 Ubiquitination.

doi: 10.1002/advs.202508673

Figure Lengend Snippet: Figure 3. FOXM1 deficiency resulted in mitochondrial dysfunction in cardiomyocytes. A) GO term enrichment analysis of downregulated proteins in cardiomyocytes isolated from Myh6+ and Foxm1fl/flMyh6+ mice based on MS data showing the top eight biological process. B) Heatmaps of mito-

Article Snippet: Foxm1 conditional knockin mouse model at the locus of ROSA26 was created by Cyagen Biosciences Inc (Guangzhou, Guangdong, China) using CRISPR/Cas-mediated genome engineering.

Techniques: Isolation

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: FOXM1 Protects Against Myocardial Ischemia-Reperfusion Injury in Rodent and Porcine Models by Suppressing MKRN1-Dependent LKB1 Ubiquitination.

doi: 10.1002/advs.202508673

Figure Lengend Snippet: Figure 4. FOXM1 deficiency induces the ubiquitin-dependent degradation of LKB1 to downregulate AMPK signaling. A) Western blot and quantification of p-AMPK and AMPK protein levels in heart samples from Myh6+ and Foxm1fl/flMyh6+ mice (n = 3). B) Western blot and quantification of p-LKB1, LKB1, and CAMKK2 protein levels in heart samples from Myh6+ and Foxm1fl/flMyh6+ mice (n = 3). C) Western blot and quantification of p-AMPK and AMPK protein levels in cultured NVCMs transfected with OE-Foxm1 or OE-NC follow treatment of LKB1 inhibitor MDK-2275 (760 nm) (n = 3). D) qRT- PCR analyses of LKB1 mRNA levels in heart samples of Myh6+ and Foxm1fl/flMyh6+ mice (n = 4). E) ChIP-qPCR analysis detected the enrichment of FOXM1 to Lkb1 promoter region in HEK293T cells transfected with Flag-Foxm1 plasmid (n = 4). F) Western blot and quantification of p-LKB1 and LKB1 protein levels in NVCMs cultured with dimethylsulfoxide (DMSO), chloroquine (CQ, 50 μm), or MG132 (50 μm) with cycloheximide (CHX, 50 μm) for the indicated durations (n = 3). G) Western blot of p-LKB1 and LKB1 in NVCMs transfected with si-NC or si-Foxm1 before treatment of cycloheximide (CHX, 50 μm) for the indicated durations (n = 3). H) Western blot of p-LKB1 and LKB1 in NVCMs infected with adenovirus expressing Foxm1 or NC before treatment of cycloheximide (CHX, 50 μm) for the indicated durations (n = 3). I) Schematic diagram showing the proteome and ubiquitinome- based experimental design using cardiomyocytes isolated from Myh6+ and Foxm1fl/flMyh6+ mice. J) Heatmap showing combined analysis of quantitative proteome and ubiquitinome results. For all statistical plots, the data are presented as mean ± SD. (A,B,D,E) By two-tailed unpaired Student’s t-test. (C) By one-way ANOVA with Bonferroni multiple comparison test.

Article Snippet: Foxm1 conditional knockin mouse model at the locus of ROSA26 was created by Cyagen Biosciences Inc (Guangzhou, Guangdong, China) using CRISPR/Cas-mediated genome engineering.

Techniques: Ubiquitin Proteomics, Western Blot, Cell Culture, Transfection, Quantitative RT-PCR, ChIP-qPCR, Plasmid Preparation, Infection, Expressing, Isolation, Two Tailed Test, Comparison

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: FOXM1 Protects Against Myocardial Ischemia-Reperfusion Injury in Rodent and Porcine Models by Suppressing MKRN1-Dependent LKB1 Ubiquitination.

doi: 10.1002/advs.202508673

Figure Lengend Snippet: Figure 5. FOXM1 Regulates AMPK via MKRN1-Mediated LKB1 Ubiquitination. A) Heatmaps of upregulated ubiquitinating enzymes and downregulated deubiquitinating enzymes in cardiomyocytes isolated from Myh6+ and Foxm1fl/flMyh6+ mice based on MS data. B) Construction of plasmids containing predicted FOXM1 binding sites within the Mkrn1 promoter, as identified by the JASPAR database. C) ChIP-qPCR analysis detected the enrichment of FOXM1 at the Mkrn1 promoter region in AC16 cells co-transfected with mutated Mkrn1 promoter plasmids and Flag-Foxm1 plasmid (n = 5). D) Dual luciferase reporter assay in AC16 cells co-transfected with NC or Flag-Foxm1 and mutated Mkrn1 promoter-driven firefly luciferase reporter plasmids (n = 5). E) Endogenous co-immunoprecipitation of MKRN1 and LKB1 in cardiomyocytes isolated from Foxm1fl/flMyh6+ mice. F) Western blot of MKRN1, p-LKB1, and LKB1 in NVCMs cultured with DMSO, lysosome inhibitor chloroquine (CQ, 50 μm), and proteasome inhibitor MG132 (50 μm) after infecting with adenovirus expressing MKRN1 or NC (n = 3). G) Western blot of p-LKB1 and LKB1 in NVCMs transfected with si-NC or si-Mkrn1; and then, treated with CHX for the indicated durations (n = 3). H) Western blot of p-LKB1 and LKB1 in NVCMs infected with adenovirus expressing MKRN1 or NC; and then, treated with cycloheximide (CHX, 50 μm) for the indicated durations (n = 3). I) Western blot of MKRN1, p-LKB1, LKB1, p-AMPK, and AMPK in NVCMs transfected with si-NC or si-Mkrn1 (n = 3). J) Western blot of MKRN1, p-LKB1, LKB1, p-AMPK, and AMPK in NVCMs infected with adenovirus expressing MKRN1 or NC (n = 3). K) HEK293T cells were transfected with His-MKRN1, Flag-LKB1, and HA-ubiquitin and treated with MG132 (50 μm). Cellular extracts from HEK293T cells were pulled down with anti-Flag antibody; and then, analyzed by Western blot with anti-HA antibody. L) HEK293T cells were transfected with His-MKRN1, Flag-LKB1, and HA-tagged mutant ubiquitin, and treated with MG132 (50 μm). Cellular extracts from HEK293T cells were pulled down with anti-Flag antibody; and then, analyzed by Western blot with anti-HA antibody (n = 3). M) HEK293T cells were transfected with His-MKRN1, Flag tagged mutated LKB1, and HA-ubiquitin, and treated with MG132 (50 μm). Cellular extracts from HEK293T cells were pulled down with anti-Flag antibody; and then, analyzed by Western blot with anti-HA antibody (n = 3). For all statistical plots, the data are presented as mean ± SD. (C,D) By one-way ANOVA with Bonferroni multiple comparison test.

Article Snippet: Foxm1 conditional knockin mouse model at the locus of ROSA26 was created by Cyagen Biosciences Inc (Guangzhou, Guangdong, China) using CRISPR/Cas-mediated genome engineering.

Techniques: Ubiquitin Proteomics, Isolation, Binding Assay, ChIP-qPCR, Transfection, Plasmid Preparation, Luciferase, Reporter Assay, Immunoprecipitation, Western Blot, Cell Culture, Expressing, Infection, Mutagenesis, Comparison

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: FOXM1 Protects Against Myocardial Ischemia-Reperfusion Injury in Rodent and Porcine Models by Suppressing MKRN1-Dependent LKB1 Ubiquitination.

doi: 10.1002/advs.202508673

Figure Lengend Snippet: Figure 6. Deleting Mkrn1 attenuates Foxm1 deficiency-induced heart failure A) Schematic diagram showing the experimental design for the construction of Foxm1/ Mkrn1fl/flMyh6+ mice. B) Ejection fraction (EF) of Myh6+, Mkrn1fl/flMyh6+, Foxm1fl/flMyh6+, and Foxm1/Mkrn1fl/flMyh6+ mice (n = 10). C) Survival rate was estimated by Kaplan–Meier method and compared by log-rank test. D) Longitudinal morphology of hearts from Myh6+, Mkrn1fl/flMyh6+, Foxm1fl/flMyh6+, and Foxm1/Mkrn1fl/flMyh6+ mice by HE staining (n = 10, scale bar = 5 mm). E) Representative electron microscopy images and quantitative analysis of mitochondrial from cardiac tissues in Foxm1fl/flMyh6+ and Foxm1/Mkrn1fl/flMyh6+ mice (n = 10; scale bar = 5 μm in upper panel and 2 μm in lower panel). F) Immunofluorescence revealed mitochondrial morphology in cardiomyocytes isolated from Foxm1fl/flMyh6+ and Foxm1/Mkrn1fl/flMyh6+ mice (n = 10; scale bar = 10 μm in upper panel and 1 μm in lower panel). G) Representative confocal images of mitochondrial transmembrane potential (Δ𝜓m) labeled by JC-1 monomer (green, marking depolarization) and JC-1 aggregate (red, marking hyperpolarization) in cardiomyocytes isolated from Foxm1fl/flMyh6+ and Foxm1/Mkrn1fl/flMyh6+ mice (n = 10; scale bar = 20 μm). H) Seahorse real-time traces and averaged data for mitochondrial oxygen consumption rate (OCR) were measured in cardiomyocytes isolated from Foxm1fl/flMyh6+ and Foxm1/Mkrn1fl/flMyh6+

Article Snippet: Foxm1 conditional knockin mouse model at the locus of ROSA26 was created by Cyagen Biosciences Inc (Guangzhou, Guangdong, China) using CRISPR/Cas-mediated genome engineering.

Techniques: Staining, Electron Microscopy, Isolation, Labeling

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: FOXM1 Protects Against Myocardial Ischemia-Reperfusion Injury in Rodent and Porcine Models by Suppressing MKRN1-Dependent LKB1 Ubiquitination.

doi: 10.1002/advs.202508673

Figure Lengend Snippet: Figure 7. Foxm1 overexpression ameliorates I/R-induced cardiac injury in mice. A) Representative images of heart sections stained by Evans blue and TTC at 24 h after myocardial-ischemia reperfusion injury from Myh6+ and Foxm1TGMyh6+ mice. The ratio of area at risk (AAR) to left ventricle (LV) and infarct area (IA) to AAR were calculated based on Evans blue and TTC staining (n = 10). B) The serum levels of lactate dehydrogenase (LDH, U/L) of Myh6+ and Foxm1TGMyh6+ mice 24 h after I/R surgery (n = 10). C) Representative TUNEL staining and relative quantification in heart samples of Myh6+ and Foxm1TGMyh6+ mice 24 h after I/R surgery (n = 10; scale bar = 20 μm). D) Seahorse real-time traces and averaged data for mitochondrial oxygen consumption rate (OCR) were measured in cardiomyocytes isolated from Myh6+ and Foxm1TGMyh6+ mice 24 h after I/R surgery using Seahorse XFe24 Analyzer (n = 10). E) Representative electron microscopy images and quantitative analysis of mitochondria from cardiac tissues in Myh6+ and Foxm1TGMyh6+ mice 24 h after I/R surgery (n = 10; scale bar = 5 μm in upper panel and 2 μm in lower panel). F) Immunofluorescence revealed mitochondrial morphology in cardiomyocytes isolated from Myh6+ and Foxm1TGMyh6+ mice 24 h after I/R surgery (n = 10; scale bar = 10 μm in upper panel and 1 μm in lower panel). G) Representative confocal images of mitochondrial transmembrane potential (Δ𝜓m) labeled by JC-1 monomer (green, marking depolarization) and JC-1 aggregate (red, marking hyperpolarization) in cardiomyocytes isolated from Myh6+ and Foxm1TGMyh6+ mice 24 h after I/R surgery (n = 10; scale bar = 20 μm). H) Western blot and quantification of MKRN1, p-AMPK, and AMPK protein levels in cardiomyocytes isolated from Myh6+ and Foxm1TGMyh6+ mice 24 h after I/R surgery (n = 3). I) Ejection fraction (EF) of Myh6+ and Foxm1TGMyh6+ mice 4 weeks after I/R surgery (n = 10). J) Picrosirius red staining of heart sections of Myh6+ and Foxm1TGMyh6+ mice 4 weeks after I/R surgery (n = 10; scale bar = 5 mm). For all statistical plots, the data are presented as mean ± SD. (A,C,D,H–J) By two-tailed unpaired Student’s t-test. (B) By Welch’s ANOVA with Dunnett’s T3 multiple comparisons test. (E–G) By Welch’s t-test.

Article Snippet: Foxm1 conditional knockin mouse model at the locus of ROSA26 was created by Cyagen Biosciences Inc (Guangzhou, Guangdong, China) using CRISPR/Cas-mediated genome engineering.

Techniques: Over Expression, Staining, TUNEL Assay, Isolation, Electron Microscopy, Labeling, Western Blot, Two Tailed Test

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: FOXM1 Protects Against Myocardial Ischemia-Reperfusion Injury in Rodent and Porcine Models by Suppressing MKRN1-Dependent LKB1 Ubiquitination.

doi: 10.1002/advs.202508673

Figure Lengend Snippet: Figure 8. Overexpressing FOXM1 mitigates I/R-induced heart failure in pigs. A) Schematic diagram illustrating experimental design of pigs. After intra- coronary injection of FOXM1-overexpressing AAV (AAV9-Foxm1) or control AAV (AAV9-NC), pigs were subjected to I/R injury (ischemia 60 min before reperfusion). The alive pigs were observed for 2 months, after which MRI examination and histological analysis for cardiac function and morphology were conducted. B) Western blot and quantification of FOXM1, MKRN1, p-AMPK, and AMPK in cardiac tissues of pigs subjected to AAV9-Foxm1 or AAV9-NC administration (n = 3). C) Representative morphology of intact hearts of pigs at 2 months after I/R surgery with AAV9-Foxm1 or AAV9-NC administration (n = 6; scale bar = 2.5 cm). D) Representative heart sections of pigs at 2 months after I/R surgery with AAV9-Foxm1 or AAV9-NC administration (n = 6;

Article Snippet: Foxm1 conditional knockin mouse model at the locus of ROSA26 was created by Cyagen Biosciences Inc (Guangzhou, Guangdong, China) using CRISPR/Cas-mediated genome engineering.

Techniques: Injection, Control, Western Blot

Journal: Oncology letters

Article Title: 7-difluoromethoxyl-5,4'-di-n-octyl genistein inhibits ovarian cancer stem cell characteristics through the downregulation of FOXM1.

doi: 10.3892/ol.2014.2080

Figure Lengend Snippet: Figure 4. Overexpression of FOXM1 attenuates the inhibitory effect of DFOG on the self‑renewal of OCSLCs derived from the SKOV3 cell line. (A) Western blot analysis showing the overexpression of FOXM1 following transfection with plasmid pcDNA3.1‑FOXM1 in the SFCs derived from the SKOV3 cells. The overexpression of FOXM1 reduced the DFOG‑induced downregulation of FOXM1, CD133, CD44 and ALDH1 expression. (B) The sphere‑forming ability of the SFCs derived from the SKOV3 cells exposed to DFOG was partly rescued following overexpression of FOXM1. Data are presented as the mean ± standard deviation (n=3). *P<0.05 vs. 0.1% DMSO. #P<0.05 vs. cells treated with the same concentration of DFOG using pcDNA3.1 transfection. DFOG, 7‑difluorome thoxyl‑5,4'‑di‑n‑octylgenistein; FOXM1, forkhead box M1; OCSLCs, ovarian cancer stem‑like cells; GEN, genistein; SFCs, sphere‑forming cells.

Article Snippet: The FOXM1 cDNA plasmid was purchased from OriGene Technologies Inc. (Rockville, MD, USA).

Techniques: Over Expression, Derivative Assay, Western Blot, Transfection, Plasmid Preparation, Expressing, Standard Deviation, Concentration Assay

Journal: Oncology letters

Article Title: 7-difluoromethoxyl-5,4'-di-n-octyl genistein inhibits ovarian cancer stem cell characteristics through the downregulation of FOXM1.

doi: 10.3892/ol.2014.2080

Figure Lengend Snippet: Figure 3. DOFG downregulates FOXM1 and CSC marker expression in OCSLCs derived from SKOV3 cells. (A) FOXM1 is highly expressed in SFCs from the SKOV3 cell line compared with the corresponding parental cells. (B) DFOG downregulated the expression of FOXM1 and CSC markers (CD133, CD44, and ALDH1) in the SFCs derived from the SKOV3 cells. DFOG, 7‑difluoromethoxyl‑5,4'‑di‑n‑octylgenistein; FOXM1, forkhead box M1; CSCs, cancer stem cells; OCSLCs, ovarian cancer stem‑like cells; GEN, genistein; SFCs, sphere‑forming cells.

Article Snippet: The FOXM1 cDNA plasmid was purchased from OriGene Technologies Inc. (Rockville, MD, USA).

Techniques: Marker, Expressing, Derivative Assay