Journal: Nature genetics

Article Title: Meningioma DNA methylation groups identify biological drivers and therapeutic vulnerabilities

doi: 10.1038/s41588-022-01061-8

Figure Lengend Snippet: a, Confocal microscopy and quantification of Annexin V in IOMM-Lee cells from Extended data figure 4e treated with actinomycin D or vehicle control for 24 hours. DNA is marked with DAPI. Scale bar 10 μM. From left to right, 96, 101, 95, 90, 98, or 75 cells are shown (ANOVA, one-sided). b, Immunoblot for Merlin, Caspase-7, cleaved Caspase-7 (cCaspase-7), or GAPDH in IOMM-Lee cells from a. c, Quantification of Annexin V confocal microscopy in MSC1 cells stably expressing sgNTC or sgNF2-2. Cells were treated as in a. From left to right, 29, 19, 40, or 30 cells are shown (ANOVA, one-sided). d, Representative images of cleaved Caspase-3 (cCaspase-3) immunohistochemistry from CH-157MN xenografts stably expressing doxycycline-inducible Merlin encoding a FLAG tag (NF2-FLAG) in NU/NU mice after 7 days of doxycycline (n=6) or vehicle treatment (n=6), and 24 hours after 4 Gy ionizing radiation (n=6) or control treatment (n=6). Scale bar 100 μM. e, Immunoblot for Merlin, IRF8, Tubulin, or Histone H3 (HH3) in cytoplasmic or nuclear fractions of M10GdCas9-KRAB cells from Extended data figure 4b. f, Normalized proteomic proximity-labeling mass spectrometry from M10G cells stably expressing Merlin constructs with APEX tags. From left to right, 2 or 3 biological replicates are shown. g, Immunoblot for IRF8 or FLAG after FLAG immunoprecipitation from M10G cells stably expressing Merlin encoding a FLAG tag (NF2FLAG). EV, empty vector. h, QPCR for the glucocorticoid receptor (NR3C1) in IOMM-Lee cells expressing a non-targeting control siRNA (siNTC) or siRNAs suppressing NR3C1 (siNR3C1). 3 biological replicates per condition (Student’s t test, one-sided). Lines represent means, and error bars represent standard error of the means.

Article Snippet: Gene over-expression For transient over-expression of FOXM1 or USF1, M10G cells were transfected with pCMV6-FOXM1 (#RC202246, Origene) or pCMV6-USF1 (#RC204915, Origene) plasmids at a ratio of 1 μg of DNA to 2 μl of FuGENE transfection reagent (#E2311, Promega).

Techniques: Confocal Microscopy, Control, Western Blot, Stable Transfection, Expressing, Immunohistochemistry, FLAG-tag, Labeling, Mass Spectrometry, Construct, Immunoprecipitation, Plasmid Preparation

Journal: Nature genetics

Article Title: Meningioma DNA methylation groups identify biological drivers and therapeutic vulnerabilities

doi: 10.1038/s41588-022-01061-8

Figure Lengend Snippet: a, Ki-67 labeling index from meningioma clinical pathology reports (n=206) across DNA methylation groups (ANOVA, one-sided). b, Representative images of meningioma Ki-67 and FOXM1 immunohistochemistry (n=92) across meningioma DNA methylation groups. Scale bar 10 μM. c, Meningioma DNA methylation analysis of chromosome 9p segment copy number deletions of any size containing the entire CDKN2A/B locus across Merlin-intact (n=8 of 192 meningiomas, 4%), Immune-enriched (n=5 of 216 meningiomas, 2%), and Hypermitotic (n=24 of 157 meningiomas, 15%) DNA methylation groups (n=565, Chi-squared test, two-sided). d, Meningioma DNA methylation (n=565) of CDKN2A (cg26349275) or CDKN2B (cg08390209) across DNA methylation groups (ANOVA, one-sided). e, tSNE plot of meningioma and meningioma cell line DNA methylation profiles. Four representative meningiomas from each DNA methylation group are shown. Triplicate meningioma M10GdCas9-KRAB cultures stably expressing a non-targeting control single-guide RNA (sgNTC) or single-guide RNAs suppressing NF2 (sgNF2), CDKN2A (sgCDKN2A), or CDKN2B (sgCDKN2B) are shown. Differences in DNA methylation groups are captured in tSNE1, and a positive shift from Immune-enriched meningiomas to Hypermitotic meningiomas mimics the shift from M10GdCas9-KRAB-sgNTC and M10GdCas9-KRAB-sgNF2 cells to M10GdCas9-KRAB-sgCDKN2A and M10GdCas9-KRAB-sgCDKN2B cells. Differences between tumors and cell lines, such as the tumor microenvironment, are captured in tSNE2. f, Meningioma DNA methylation analysis of chromosome 1p segment copy number amplifications of any size containing the entire USF1 locus across Merlin-intact (n=0 of 192 meningiomas, 0%), Immune-enriched (n=2 of 216 meningiomas, 4%), and Hypermitotic (n=38 of 157 meningiomas, 24%) DNA methylation groups (n=565, Chi-squared test, two-sided). g, USF1 ChIP-QPCR in DI98 meningioma cells for the CDK6 promoter compared to negative control primers targeting a gene desert (NC1) or a gene not predicted to be bound by USF1 (NC2) from ChIP sequencing. **p=0.001 (Student’s t test, one-sided, no adjustment for multiple comparisons). h, QPCR for CDK6 in M10GdCas9-KRAB cells expressing sgNTC or a single-guide RNA suppressing USF1 (sgUSF1), or M10G cells over-expressing USF1 or empty vector (EV). *p=0.003, **p=0.001 (Student’s t test, one-sided, no adjustment for multiple comparisons). i, Relative colony area of CH-157MN cells stably over-expressing USF1 or EV after 10 days of clonogenic growth. **p=0.001 (Student’s t test, one-sided, no adjustment for multiple comparisons). Lines represent means, and error bars represent standard error of the means.

Article Snippet: Gene over-expression For transient over-expression of FOXM1 or USF1, M10G cells were transfected with pCMV6-FOXM1 (#RC202246, Origene) or pCMV6-USF1 (#RC204915, Origene) plasmids at a ratio of 1 μg of DNA to 2 μl of FuGENE transfection reagent (#E2311, Promega).

Techniques: Labeling, DNA Methylation Assay, Immunohistochemistry, Stable Transfection, Expressing, Control, ChIP-qPCR, Negative Control, ChIP-sequencing, Plasmid Preparation

Journal: Nature genetics

Article Title: Meningioma DNA methylation groups identify biological drivers and therapeutic vulnerabilities

doi: 10.1038/s41588-022-01061-8

Figure Lengend Snippet: a, Predicted network of FOXM1-regulated pathways in Hypermitotic meningiomas based on H3K27ac ChIP sequencing of 25 meningiomas with matched RNA sequencing and DNA methylation profiling (15 Hypermitotic, 10 non-Hypermitotic). b, Immunoblot for Merlin, FOXM1, or GAPDH in IOMM-Lee meningioma cells stably expressing a non-targeting ontrol shRNA (shNTC) or shRNAs suppressing NF2 (shNF2-1 or shNF2-2), after treatment with actinomycin D or vehicle control for 24 hours. c, QPCR for FOXM1 in M10G meningioma cells over-expressing FOXM1 or empty vector (EV). 3 biological replicates per condition. ***p≤0.0001 (Student’s t test, one-sided). d, Quantification of Annexin V confocal microscopy in M10G cells over-expressing FOXM1 or EV after treatment with actinomycin D or vehicle control for 24 hours. From left to right, 57, 58, 65, or 60 cells are shown (ANOVA, one-sided). Lines represent means, and error bars represent standard error of the means.

Article Snippet: Gene over-expression For transient over-expression of FOXM1 or USF1, M10G cells were transfected with pCMV6-FOXM1 (#RC202246, Origene) or pCMV6-USF1 (#RC204915, Origene) plasmids at a ratio of 1 μg of DNA to 2 μl of FuGENE transfection reagent (#E2311, Promega).

Techniques: ChIP-sequencing, RNA Sequencing, DNA Methylation Assay, Western Blot, Stable Transfection, Expressing, shRNA, Control, Plasmid Preparation, Confocal Microscopy

Journal: Nature genetics

Article Title: Meningioma DNA methylation groups identify biological drivers and therapeutic vulnerabilities

doi: 10.1038/s41588-022-01061-8

Figure Lengend Snippet: a, Relative colony area of M10G, BenMen, CH-157MN, or IOMM-Lee meningioma cells after 10 days of clonogenic growth and treatment with abemaciclib, ribociclib, or palbociclib. 3 biological replicates per condition per timepoint. b, Relative colony area of M10GdCas9-KRAB cells expressing sgNTC, sgCDKN2A, or sgCDKN2B after 10 days of clonogenic growth and treatment with abemaciclib. 3 biological replicates per condition. *p=0.002, **p=0.001 (Student’s t test, one-sided). Data are normalized to growth with vehicle treatment of each cell lines. c, Relative colony area of CH-157MN cells stably over-expressing USF or empty vector (EV) after 10 days of clonogenic growth and treatment with abemaciclib. 3 biological replicates per condition. **p=0.001 (Student’s t test, one-sided). Data are normalized to growth with vehicle treatment of each cell lines. d, Quantification of BenMen peri-organoid intensity after 10 days of growth and treatment with abemaciclib or vehicle control Representative images of meningioma (red) and organoid (green) cells are shown. Scale bar 100 μM. 5 biological replicates per condition (ANOVA, one-sided). e, Representative immunoblots from CH-157MN xenografts in NU/NU mice (left) harvested at intervals after a single treatment of abemaciclib (100 μg/g) via oral gavage (right). f, Representative images of CH-157MN xenograft Ki-67 immunohistochemistry after a daily treatment of abemaciclib or control. Scale bar 1 mm. Lines represent means, and error bars represent standard error of the means.

Article Snippet: Gene over-expression For transient over-expression of FOXM1 or USF1, M10G cells were transfected with pCMV6-FOXM1 (#RC202246, Origene) or pCMV6-USF1 (#RC204915, Origene) plasmids at a ratio of 1 μg of DNA to 2 μl of FuGENE transfection reagent (#E2311, Promega).

Techniques: Inhibition, Expressing, Stable Transfection, Plasmid Preparation, Control, Western Blot, Immunohistochemistry

Journal: Bioengineered

Article Title: Downregulation of sperm-associated antigen 5 inhibits melanoma progression by regulating forkhead box protein M1/A disintegrin and metalloproteinase 17/NOTCH1 signaling.

doi: 10.1080/21655979.2022.2031670

Figure Lengend Snippet: Figure 5. Downregulation of SPAG5 decreased the expression of ADAM17, HES1 and NOTCH1 through downregulation of FOXM1 in MM. (a) SPAG5 downregulation on the expression of FOXM1 was determined by Western blotting. (b)The expression of FOXM1, ADAM17, HES1 and NOTCH1 were detected by Western blotting in A375 and A2058 cells with FOXM1 overexpression or without. (c) Western blotting was used to detect the expression of FOXM1, ADAM17, NOTCH1 and HES1 in A375 and A2058 cells transfected with shNC+NC, shSPAG5-2+ NC and shSPAG5-2+ FOXM1. (n = 3, *P < 0.05, **P < 0.01; NC, negative control).

Article Snippet: To upregulate FOXM1 and ADAM17 expression, MM cells were transiently transfected with the overexpression plasmids of FOXM1 (cat no. RC202246, Origene, Beijing, China) and ADAM17 (cat no. SC316426, Origene) with the help of Lipofectamin 2000 (Invitrogen, USA) according to the manufacturer’s instructions.

Techniques: Expressing, Western Blot, Over Expression, Transfection, Negative Control

Journal: Bioengineered

Article Title: Downregulation of sperm-associated antigen 5 inhibits melanoma progression by regulating forkhead box protein M1/A disintegrin and metalloproteinase 17/NOTCH1 signaling.

doi: 10.1080/21655979.2022.2031670

Figure Lengend Snippet: Figure 6. Downregulation of SPAG5 weakened the in vivo tumor formation ability of A375 cells. (a) Tumor volume and weight. (b) The expression of SPAG5, FOXM1, ADAM17, NOTCH1, E-cadherin and N-cadherin in shNC and shSPAG5-2 groups were detected using IHC. (c) Hematoxylin eosin staining was used to assess the lung metastasis of MM (Each group had 5 mice, **P < 0.01).

Article Snippet: To upregulate FOXM1 and ADAM17 expression, MM cells were transiently transfected with the overexpression plasmids of FOXM1 (cat no. RC202246, Origene, Beijing, China) and ADAM17 (cat no. SC316426, Origene) with the help of Lipofectamin 2000 (Invitrogen, USA) according to the manufacturer’s instructions.

Techniques: In Vivo, Expressing, Staining

Journal: STAR Protocols

Article Title: Protocol for quantifying drug sensitivity in 3D patient-derived ovarian cancer models

doi: 10.1016/j.xpro.2024.103274

Figure Lengend Snippet:

Article Snippet: CD326 (EpCAM) antibody, anti-human, PE, REAfinity (flow cytometry 1:200) , Miltenyi Biotec , Cat# 130-110-999; RRID: AB_2657495.

Techniques: Immunofluorescence, Flow Cytometry, Control, Recombinant, Red Blood Cell Lysis, Saline, Software, Cell Culture, Plasmid Preparation, Microscopy

Journal: PLoS ONE

Article Title: A FOXM1 Dependent Mesenchymal-Epithelial Transition in Retinal Pigment Epithelium Cells

doi: 10.1371/journal.pone.0130379

Figure Lengend Snippet: A. Immunocytochemistry was performed for CRALBP, ZO1, αSMA and Ki67 at Day 0 (D0), Day 3 (D3) and Day 35 (D35). Arrowheads point towards Ki67 positive nuclei. B. Microarray heatmap of the expression profiles of the top 250 genes, ranked by the significance of their expression changes, over time in culture. Raw expression data are mean centred and scaled to unit variance prior to clustering. A schematic of the scaled expression is shown on the right where individual gene profiles are in light grey and the mean expression profile is shown in black. C. Microarray heatmap showing transcript expression for a panel of representative markers over a timecourse of RPE culture. D. Immunocytochemistry for FOXM1 at Day 2 and Day 14 of RPE culture. Arrowheads point towards FOXM1 positive nuclei. E. Quantification of immunocytochemistry showing percentage of nuclei staining positive for FOXM1 over time. Bars represent Mean ± SD (n = 3). F. Expression of FOXM1 transcript measured using qPCR (relative to housekeeping genes ACTB and GAPDH ) in iPSC derived RPE, human foetal RPE and ARPE19 cells over time. Bars represent Mean ± SD (n = 3)

Article Snippet: The FOXM1 overexpression vector was purchased from Origene (SC112825) and transfected into RPE using the Effectene reagent (Qiagen) according to manufacturer’s instructions, although some effects of reagent toxicity were noted.

Techniques: Immunocytochemistry, Microarray, Expressing, Staining, Derivative Assay

Journal: PLoS ONE

Article Title: A FOXM1 Dependent Mesenchymal-Epithelial Transition in Retinal Pigment Epithelium Cells

doi: 10.1371/journal.pone.0130379

Figure Lengend Snippet: A. qPCR based measurement of transcript expression of a panel of epithelial (red) and mesenchymal (green) markers at Day 10 post siFOXM1 transfection (except levels of FOXM1 itself which are measured at Day 2 post knockdown). Data is normalized to transfection with non-targeting siRNA used as a control. ACTB , GAPDH , IPO8 and HPRT1 are used as housekeeping genes. Bars represent Mean + SD (n = 3). P<0.05 (Student’s t-test). B. Immunocytochemistry for PMEL17 upon FOXM1 knockdown (siFOXM1) or overexpression (pFOXM1) at Day 10 post transfection. C. Level of knockdown obtained upon transient transfection of siRNA against SNAI2, SNAI1, ZEB1, TWIST1 and GSC. Knockdown was measured by qPCR at Day 6 post transfection and is expressed relative to non-targeting siRNA used as control. CYC1 and GAPDH were used as housekeeping genes. Bars represent Mean ± SD (n = 6–9). Knockdown of EMT-TF expression was significant, P<0.05 (Student’s t-test). D. No significant effect on PMEL , MITF or BEST1 expression was observed under the same conditions described above for Fig 2C.

Article Snippet: The FOXM1 overexpression vector was purchased from Origene (SC112825) and transfected into RPE using the Effectene reagent (Qiagen) according to manufacturer’s instructions, although some effects of reagent toxicity were noted.

Techniques: Expressing, Transfection, Knockdown, Control, Immunocytochemistry, Over Expression

Journal: PLoS ONE

Article Title: A FOXM1 Dependent Mesenchymal-Epithelial Transition in Retinal Pigment Epithelium Cells

doi: 10.1371/journal.pone.0130379

Figure Lengend Snippet: A. Graph showing quantification of immunocytochemistry where % Ki67 (n = 3) or % EdU (n = 6) is plotted on the left Y axis and relative expression of FOXM1 transcript (n = 3; ACTB used as housekeeping gene) on the right Y axis over days in culture (x axis). B. Quantification of change in FOXM1 transcript upon transient overexpression (pFOXM1) or knockdown (siFOXM1), 48h post transfection, measured by qPCR. Data is normalized to appropriate controls (Empty vector for pFOXM1 and non-targeting siRNA for siFOXM1). Bars represent Mean + SD (n = 3). C. Quantification of change in EdU incorporation upon FOXM1 overexpression or knockdown, 72h post transfection. Data is normalized to appropriate controls (Empty vector for pFOXM1 and non-targeting siRNA for siFOXM1). Bars represent Mean + SD (n = 4). P<0.0001 (Student’s t-test). D. Quantification of immunocytochemistry for Ki67 upon siRNA mediated knockdown of non-targeting control, GAPDH, SNAI1, SNAI2 and FOXM1, at Day 6 post transfection. Bars represent Mean + SD (n = 3). n.s non-significant, * p<0.05 Student’s t-test. E. Effect of Thiostrepton on EdU incorporation [left Y axis, red] and FOXM1 transcript expression measured by qPCR [right Y axis, blue]. Bars represent Mean ± SD (n = 6). F. Bright-field microscopy showing a scratch introduced in a RPE monolayer at 0 hrs and 19hrs in the presence of DMSO or 10μM Thiostrepton. Edge of the scratch is marked with a white line. Scale bar = 200 μm. G. Quantification of F (above). Bars represent Mean + SD (n = 7). P<0.0001 (Student’s t-test)

Article Snippet: The FOXM1 overexpression vector was purchased from Origene (SC112825) and transfected into RPE using the Effectene reagent (Qiagen) according to manufacturer’s instructions, although some effects of reagent toxicity were noted.

Techniques: Immunocytochemistry, Expressing, Over Expression, Knockdown, Transfection, Plasmid Preparation, Control, Microscopy

Journal: PLoS ONE

Article Title: A FOXM1 Dependent Mesenchymal-Epithelial Transition in Retinal Pigment Epithelium Cells

doi: 10.1371/journal.pone.0130379

Figure Lengend Snippet: A. Percentage of FOXM1 peaks within proximity boundaries to Transcription Start Sites (TSS). B. Plot showing the mean read depth over FOXM1 peaks with a majority of binding within 100bp of peak centres. C. Protein coding genes with a FOXM1 peak within 1kb of TSS are highly enriched for GO categories relevant to cell cycle related functions but not EMT, MET, epithelial or mesenchymal related functions. In addition to the GO category, enrichment was also tested at a published EMT gene signature with no significant binding seen. Enrichment was calculated using a hypergeometric distribution, the—log 10 p-value is shown. Dashed line represents p = 0.05. D. Schematic showing FOXM1 binding to the promoters of representative cell cycle genes; CDK12, CDC20, CDC5L & CDKN1A. ChIP-seq coverage is shown in blue and annotated genomic features shown in orange. E. Quantification of change in transcript expression of representative FOXM1 bound genes, measured by qPCR, upon siRNA mediated FOXM1 knockdown (relative to transfection with non-targeting siRNA used as a control), 72h post transfection. ACTB is used as a housekeeping gene. Bars represent Mean + SD (n = 3). P<0.05 (Student’s t-test). F. Significantly enriched transcription factor motifs in FOXM1 peaks alongside frequencies of occurrence.

Article Snippet: The FOXM1 overexpression vector was purchased from Origene (SC112825) and transfected into RPE using the Effectene reagent (Qiagen) according to manufacturer’s instructions, although some effects of reagent toxicity were noted.

Techniques: Binding Assay, ChIP-sequencing, Expressing, Knockdown, Transfection, Control

Journal: PLoS ONE

Article Title: A FOXM1 Dependent Mesenchymal-Epithelial Transition in Retinal Pigment Epithelium Cells

doi: 10.1371/journal.pone.0130379

Figure Lengend Snippet: A. Quantification of change in cell density (number of DAPI positive nuclei per cm 2 imaged area) upon FOXM1 overexpression or knockdown, 72h post transfection. Data is normalized to appropriate controls (Empty vector for pFOXM1 and non-targeting siRNA for siFOXM1). Bars represent Mean + SD (n = 4). P<0.0001 (Student’s t-test). B. Heatmap showing changes in gene expression of a panel of representative markers over a timecourse of RPE culture where cells are seeded at high (100000 cells/cm 2 ) or low (8000 cells/cm 2 ) density. C. Plot showing differential expression of BMP7 and Wnt5B transcripts extrapolated from the microarray data. The shaded area represents 95% confidence intervals around the point estimates (circles) of the difference between the mean high density expression vs the mean low density expression.

Article Snippet: The FOXM1 overexpression vector was purchased from Origene (SC112825) and transfected into RPE using the Effectene reagent (Qiagen) according to manufacturer’s instructions, although some effects of reagent toxicity were noted.

Techniques: Over Expression, Knockdown, Transfection, Plasmid Preparation, Gene Expression, Quantitative Proteomics, Microarray, Expressing

Journal: PLoS ONE

Article Title: A FOXM1 Dependent Mesenchymal-Epithelial Transition in Retinal Pigment Epithelium Cells

doi: 10.1371/journal.pone.0130379

Figure Lengend Snippet: RPE first acquire a mesenchymal morphology upon dissociation and culture followed by proliferation and mesenchymal-epithelial transition to re-uptake an epithelial phenotype. Proliferation of RPE is directly regulated by FOXM1 which also affects expression of BMP7 and Wnt5B by an unknown mechanism. Both these activities are required for successful MET and epithelialization.

Article Snippet: The FOXM1 overexpression vector was purchased from Origene (SC112825) and transfected into RPE using the Effectene reagent (Qiagen) according to manufacturer’s instructions, although some effects of reagent toxicity were noted.

Techniques: Expressing

Journal: The American journal of pathology

Article Title: Genome-wide expression analysis of Middle Eastern colorectal cancer reveals FOXM1 as a novel target for cancer therapy.

doi: 10.1016/j.ajpath.2010.10.020

Figure Lengend Snippet: Figure 2. Tissue microarray based immunohistochemical analysis of FoxM1, MMP-9, and Ki-67 in CRC patients. A CRC array spot showing overexpression of FoxM1 (A), MMP-9 (C), and Ki-67 (E). CRC tissue array spots showing low expression of FoxM1 (B), MMP-9 (D), and Ki-67 (F). All images were taken on an Olympus BX 51 microscope, 20 0.70 objective (inset: magnified view, 40 0.85). G: Box-plots indicate the mean and SD of FoxM1 expres- sion in three groups: adjacent normal colorectal mucosa, adenoma, and CRC. FoxM1 expression in colorectal adenomas (49.25 37.91) and CRCs (mean 53.26 39.51) was greater than in normal colon (1.51 5.02) (P 0.001 and P 0.001, respectively); however, there was no difference in expression between CRC and adenomas (P 0.6507). The asterisks indicate the wide range of FoxM1 expression in the normal colorectal mucosa group. FoxM1 expression in normal colorectal mucosa was in the range of 0 to 5 in 95% of the samples; however, a few samples (4%) showed expression score of 10, and 2 samples had extreme scores of 20 and 70. In the cancer group, the letter O indicates one CRC spot with an IHC score of 190, and it indicates extreme deviation from the mean.

Article Snippet: Human cDNA FoxM1 clone (catalog no. SC128214) vector alone (pCMV6-XL5) was purchased from OriGene Technologies (Rockville, MD).

Techniques: Microarray, Immunohistochemical staining, Over Expression, Expressing, Microscopy

Journal: The American journal of pathology

Article Title: Genome-wide expression analysis of Middle Eastern colorectal cancer reveals FOXM1 as a novel target for cancer therapy.

doi: 10.1016/j.ajpath.2010.10.020

Figure Lengend Snippet: Figure 3. FoxM1 expression and its effect on cell growth in colorectal cancer (CRC) cell lines. A: Upper panel: LOVO, Cl-11, COLO-320, CX1, DLD-1, and HCT-15 cells were lysed and pro- teins were immunoblotted with FoxM1 and -actin. Lower panel: The data obtained from the immunoblot analyses of FoxM1 were used to evaluate relative expression by spot densitome- try. B: CRC cells were incubated with 5 and 10 mol/L thiostrepton for 48 hours. Cell prolifer- ation assays were performed using MTT as de- scribed in Materials and Methods. Data are re- ported as means SD of three independent experiments, with replicates of six wells for all of the doses and vehicle control for each experi- ment. *P 0.05, Student’s t-test. C: Thiostrep- ton-induced increase in subG1/Apo fraction of CRC cells. LOVO, Colo-320, DLD-1, and HCT-15 CRC cells were treated with 5 and 10 mol/L thiostrepton for 48 hours. Thereafter, the cells were washed, fixed, and stained with propidium iodide and were analyzed for DNA content by flow cytometry as described in Materials and Methods. At least three independent experiments were performed for all of the cell lines. *P 0.05, Student’s t-test. D: Thiostrepton-me- diated apoptosis in CRC cell lines.LOVO, COLO- 320, DLD-1, and HCT-15 CRC cells were treated with 5 and 10 mol/L thiostrepton for 48 hours and cells were subsequently stained with fluo- rescein-conjugated annexin-V and propidium io- dide and analyzed by flow cytometry. Right lower (RL) quadrant denotes annexin v postive cells depicting alive apoptotic cells while right upper (RU) quadrant denotes annexin V and propidium iodide positive cells depicting dead apoptotic cells. Apoptosis is measured as the sum of RL and RU. X-axis denotes number of annexin V positive cells and y-axis denotes num- ber of propidium iodide positive cells.

Article Snippet: Human cDNA FoxM1 clone (catalog no. SC128214) vector alone (pCMV6-XL5) was purchased from OriGene Technologies (Rockville, MD).

Techniques: Expressing, Western Blot, Incubation, Control, Staining, Flow Cytometry

Journal: The American journal of pathology

Article Title: Genome-wide expression analysis of Middle Eastern colorectal cancer reveals FOXM1 as a novel target for cancer therapy.

doi: 10.1016/j.ajpath.2010.10.020

Figure Lengend Snippet: Figure 4. Overexpression of FoxM1 promoted growth and clonogenicity and induced invasion and migration in CRC cells. A: FoxM1 cDNA was transfected in HCT-15 and LOVO and stable clones were selected. HCT-15 and LOVO cells with vector alone or FoxM1-transfected clones were lysed and proteins were immunoblotted with FoxM1 and -actin. B: Vector and FoxM1- overexpressing HCT-15 clones 1 and 3 and LOVO clones 1 and 2 were seeded in 96-well plates. After 48 hours, cell proliferation assays were performed using MTT as described in Mate- rials and Methods. Data are reported as means SD of three independent experiments, with repli- cates of six wells for all of the doses and vehicle control for each experiment *P 0.05, Student’s t-test. C: Clonogenic assays were performed using vector and FoxM1-overexpressing HCT-15 clones 1 and 3 as described in Materials and Methods (upper panel). HCT-15 vector and HCT-15 clones 1 and 3 were plated in soft agar plates for 4 weeks, after which cells were stained and man- ually counted (lower panel). *P 0.05, Stu- dent’s t-test. D: HCT-15 and LOVO cells with vector alone or FoxM1-transfected clones were lysed and proteins were immunoblotted with MMP-2, MMP-9, and -actin. E: Invasion-migra- tion assays were performed using vector and FoxM1-overexpressing HCT-15 clones 1 and 3 as described in Materials and Methods. F: Manual counts of invasion and migration experiment of HCT-15 vector, clone 1, and clone 3 cells treated as just described for (E). *P 0.005.

Article Snippet: Human cDNA FoxM1 clone (catalog no. SC128214) vector alone (pCMV6-XL5) was purchased from OriGene Technologies (Rockville, MD).

Techniques: Over Expression, Migration, Transfection, Clone Assay, Plasmid Preparation, Control, Staining

Journal: The American journal of pathology

Article Title: Genome-wide expression analysis of Middle Eastern colorectal cancer reveals FOXM1 as a novel target for cancer therapy.

doi: 10.1016/j.ajpath.2010.10.020

Figure Lengend Snippet: Figure 5. Overexpression of FoxM1 protects HCT-15 cells from thiostrepton-mediated inhibi- tion, apoptosis, and inhibition of migration and invasion. A: Vector and FoxM1-transfected HCT-15 clones 1 and 3 were treated with 5 and 10 mol/L thiostrepton for 48 hours. Cell proliferation assays were performed using MTT as described in Mate- rials and Methods. Data are reported as means SD of three independent experiments, with repli- cates of six wells for all of the doses and vehicle control for each experiment. *P 0.05, Student’s t-test. B: Vector and FoxM1-transfected HCT-15 cells were treated with 5 and 10 mol/L thiostrep- ton for 48 hours. Thereafter, the cells were washed, fixed, and stained with propidium iodide, and analyzed for DNA content by flow cytometry. At least three independent experiments were per- formed for all of the cell lines. *P 0.05, Student’s t-test. C: After treatment with 5 and 10 mol/L thiostrepton for 48 hours, cells were lysed and proteins were separated on SDS-polyacrylamide gel electrophoresis and were immunoblotted with caspase-3, cleaved caspase-3, PARP, and -actin. D: HCT-15 vector and HCT-15 clone 3 cells were transfected with scrambled siRNA and FoxM1 siRNA (100 nmol/L). After 48 hours, cells were lysed and proteins were immunoblotted with an- tibodies against caspase-3, cleaved caspase-3, PARP, and -actin. HCT-15 vector and FoxM1-trans- fected HCT-15 clone 3 were treated with 5 and 10 mol/L thiostrepton for 48 hours and activity of VEGF (E) and of MMP-2 (F) was determined by enzyme-linked immunosorbent assay. *P 0.05, Stu- dent’s t-test. G: HCT-15 vector and FoxM1-transfected HCT-15 clone 3 cells were treated with 5 and 10 mol/L thiostrepton and invasion and migration as- says were performed. H: After treatment with 5 and 10 mol/L thiostrepton for 48 hours, cells were lysed and proteins were separated on SDS-PAGE and im- munoblotted with FoxM1, MMP-2, MMP-9, and - actin.

Article Snippet: Human cDNA FoxM1 clone (catalog no. SC128214) vector alone (pCMV6-XL5) was purchased from OriGene Technologies (Rockville, MD).

Techniques: Over Expression, Inhibition, Migration, Plasmid Preparation, Transfection, Clone Assay, Control, Staining, Flow Cytometry, Polyacrylamide Gel Electrophoresis, Activity Assay, Enzyme-linked Immunosorbent Assay, SDS Page

Journal: The American journal of pathology

Article Title: Genome-wide expression analysis of Middle Eastern colorectal cancer reveals FOXM1 as a novel target for cancer therapy.

doi: 10.1016/j.ajpath.2010.10.020

Figure Lengend Snippet: Figure 6. FoxM1 overexpression promotes colorectal cancer tumor growth. Nude mice were injected subcutaneously with HCT-15 vec- tor and HCT-15 clone 1 and clone 3 cells. A: The volume of each tumor was measured every week. The average (n 6) tumor volume in each group of mice was calculated, *P 0.05. B: After 6 weeks of treatment, mice were sacrificed and the mean tumor weight (SD) was calcu- lated in each group. *P 0.05, Student’s t-test. C: Representative tumor images of each group of mice after necropsy. Insets show 10 magnifi- cation. D: Whole-cell homogenates from mice injected with HCT-15 vector and HCT-15 clone 1 and clone 3 were immunoblotted with antibod- ies against FoxM1, MMP-2, MMP-9, and -actin.

Article Snippet: Human cDNA FoxM1 clone (catalog no. SC128214) vector alone (pCMV6-XL5) was purchased from OriGene Technologies (Rockville, MD).

Techniques: Over Expression, Injection, Plasmid Preparation

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: The FoxM1 cDNA plasmid was purchased from OriGene Technologies Inc. (Rockville, MD, 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: The FoxM1 cDNA plasmid was purchased from OriGene Technologies Inc. (Rockville, MD, 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: The FoxM1 cDNA plasmid was purchased from OriGene Technologies Inc. (Rockville, MD, 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: The FoxM1 cDNA plasmid was purchased from OriGene Technologies Inc. (Rockville, MD, USA).

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