Journal: The FASEB Journal

Article Title: Circular RNA circBFAR promotes glioblastoma progression by regulating a miR‐548b/FoxM1 axis

doi: 10.1096/fj.202101307r

Figure Lengend Snippet: FIGURE 6 FoxM1 is a direct target of miR-548b. (A) Schematic of the selection showed the intersection between miR-548b potential targets predicted by 5 databases (FC > 2; FDR < 0.01). (B) Levels of FoxM1 following transfection of miR-548b/miR-NC or miR-548b inhibitor/inhibitor NC in U251 and LN229 cells. (C) Binding sites between FoxM1 and miR-548b. (D) Analysis of the luciferase activity between U251 cells co-transfected with miR-548b mimics/miR-NC and the luciferase reporter psi-CHECK2-FoxM1 WT/MUT. (E) Relationship between miR-548b expression and FoxM1 levels in 50 GBM patients was assessed using Pearson's correlation analysis. (F) Expression of FoxM1 was increased in GBM tissues compared with the normal brain tissues based on data obtained from TCGA. (G) Expression of FoxM1 was detected in clinical GBM tissues. n = 50. (H) Kaplan-Meier analysis suggested that FoxM1 levels were negatively associated with the prognosis of GBM based on data obtained from TCGA. (I) Expression of FoxM1 in different cell lines. Data are presented as the mean ± standard error of at least three independent experiments. *p < .05, **p < .01, ***p < .001. FC, fold change; FDR, false discovery rate; FoxM1, Forkhead Box Protein M1; GBM, glioblastoma; miRNA/miR, microRNA; NC, negative control; siRNA, short interfering RNA; TCGA, The Cancer Genome Atlas

Article Snippet: Protein concentrations were detected using the BCA protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China), and the protein (40 μg) was separated by 10% SDS- PAGE and transferred onto a PVDF membrane (Beyotime Institute of Biotechnology, Shanghai, China) and added with the primary antibodies overnight at 4°C: rabbit anti- FoxM1 (Rabbit polyclonal antibody, 1:1000; Cell Signaling Technology, Danvers, MA, USA), and mouse antiGAPDH (Mouse Monoclonal Antibody, 1:1000; Santa Cruz Biotechnology, Inc., USA) and then with secondary antibodies for 1 h. All the bands were conducted using ECL detection with a ChemiDocTM MP Imaging System (Bio- Rad Laboratories, Inc.).

Techniques: Selection, Transfection, Binding Assay, Luciferase, Activity Assay, Expressing, Negative Control, Small Interfering RNA

Journal: The FASEB Journal

Article Title: Circular RNA circBFAR promotes glioblastoma progression by regulating a miR‐548b/FoxM1 axis

doi: 10.1096/fj.202101307r

Figure Lengend Snippet: FIGURE 7 circBFAR promotes cell proliferation by modulating a miR-548b/FOXM1 axis. (A) Pearson's correlation analysis shows the correlation between circBFAR and FoxM1 levels in clinical GBM patients (n = 50). (B and C) circBFAR suppression decreased FoxM1 expression both at the (B) mRNA and (C) protein expression levels, whereas the miR-548b inhibitor impeded the level of FoxM1. (D) Cell Counting Kit-8 assays showed the proliferation of both U251 and LN229 cells following transfection with si-circBFAR/si-NC, miR-548b inhibitor/inhibitor NC, either alone or in combination. (E) Transwell invasion assays of both U251 and LN229 cells. Magnification, ×100; scale bar, 50 µm. Data are presented as the mean ± standard error of at least three independent experiments. *p < .05, **p < .01, ***p < .001. FoxM1, Forkhead Box Protein M1; NC, negative control; siRNA, short interfering RNA

Article Snippet: Protein concentrations were detected using the BCA protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China), and the protein (40 μg) was separated by 10% SDS- PAGE and transferred onto a PVDF membrane (Beyotime Institute of Biotechnology, Shanghai, China) and added with the primary antibodies overnight at 4°C: rabbit anti- FoxM1 (Rabbit polyclonal antibody, 1:1000; Cell Signaling Technology, Danvers, MA, USA), and mouse antiGAPDH (Mouse Monoclonal Antibody, 1:1000; Santa Cruz Biotechnology, Inc., USA) and then with secondary antibodies for 1 h. All the bands were conducted using ECL detection with a ChemiDocTM MP Imaging System (Bio- Rad Laboratories, Inc.).

Techniques: Expressing, Cell Counting, Transfection, Negative Control, Small Interfering RNA

Journal: Journal of Cell Science

Article Title: APC/C Cdh1 is required for the termination of chromosomal passenger complex activity upon mitotic exit

doi: 10.1242/jcs.251314

Figure Lengend Snippet: N-terminal sequences of borealin are essential for Cdh1 binding and APC/C Cdh1 -mediated polyubiquitylation. (A) Comparison of borealin amino acid sequences of Homo sapiens , Mus musculus , Xenopus tropicalis and Drosophila melanogaster from residues 18 to 30 and 68 to 77 of the human sequence. Colored circles indicate residues involved in interactions with INCENP (green circles) and survivin (blue circles), as shown in a previous report . (B) 293T cells were co-transfected with empty vector (EV), FLAG-tagged wild-type (WT) borealin, FLAG-tagged borealin 5E mutant (L21E/F24E/L25E/F28E/V32E), FLAG-tagged borealin W70E/F74E mutant, FLAG-tagged borealin 5E+W70E/F74E mutant (L21E/F24E/L25E/F28E/V32E/W70E/F74E), or FLAG-tagged borealin deletion mutant (Δ18–77) and HA–Cdh1 and treated with 10 μM MG132 for 5 h. Cell extracts were immunoprecipitated (IP) with an affinity-purified monoclonal antibody against FLAG and analyzed by immunoblotting as indicated. Ponceau S staining is shown as a loading control. WCE: whole cell extracts. (C) An in vivo ubiquitylation assay was performed. EV, FLAG-tagged WT borealin, or mutants were co-transfected with HA-tagged ubiquitin in 293T cells. Cell extracts were immunoprecipitated using an anti-FLAG antibody and analyzed by immunoblotting as indicated. Ponceau S staining is shown as a loading control. (D) EV, FLAG-tagged WT borealin, or the 5E+W70E/F74E borealin mutant were co-transfected with HA-tagged Cdh1 in 293T cells. Cell extracts were immunoblotted with anti-HA and anti-FLAG antibodies. Vinculin expression is shown as a loading control. Densitometric analysis of FLAG–borealin and vinculin was performed. The graph shows the fold change of FLAG–borealin:vincullin ratio in HA–Cdh1 transfected cells, compared with empty vector transfected cells. (E) HeLa cells transfected with FLAG-tagged WT borealin or FLAG-tagged borealin 5E+W70E/F74E mutant were synchronized at prometaphase using nocodazole (M phase). Cells were treated with 25 μg/ml cycloheximide (CHX) at 2 h after release from nocodazole block (G1 phase) and collected at indicated time points. Cell extracts were immunoblotted for the indicated proteins. Vinculin expression is shown as a loading control. Blots in B–E are representative of two experiments.

Article Snippet: Commercial antibodies used were: mouse monoclonal anti-Aurora A, anti-Aurora B, and anti-p27 Kip1 antibodies (#610938, #611082 and #610242, respectively; BD Transduction Laboratories); rabbit polyclonal anti-INCENP (#2786; Cell Signaling Technology); rabbit monoclonal anti-FoxM1 p-T600, anti-HA, anti-Aurora A p-T288/Aurora B p-T232/Aurora C p-T198, and anti-vinculin antibodies (#14655, #3724, #2914 and #13901, respectively; Cell Signaling Technology); anti-Cdc23 and anti-INCENP antibodies (#ab72206 and #ab12183; Abcam); rabbit monoclonal anti-Aurora B (#ab45145; Abcam); mouse monoclonal anti-Cdh1/Fzr and anti-Cdc20 antibodies (#K0085-3 and #K0140-3, respectively; MBL); rabbit polyclonal anti-cyclin A antibody (#sc-751; Santa-Cruz Biotechnology); mouse monoclonal anti-cyclin B1 antibody (#sc-245; Santa-Cruz Biotechnology); anti-Cdc27 and anti-β-actin antibodies (#C7104 and #A5441, respectively; Sigma-Aldrich); mouse monoclonal anti-HA, anti-FLAG, anti-c-Myc, and anti-GFP antibodies (#014-21881, #018-22381, #011-21874 and #012-22541, respectively; Wako); rabbit polyclonal anti-survivin antibody (#NB500-201; Novus Biologicals); rabbit polyclonal anti-Cul1 antibody (#71-8700; Thermo Fisher Scientific); and rabbit polyclonal anti-H3 p-S10 antibody (#06-570; Merck Millipore).

Techniques: Binding Assay, Comparison, Sequencing, Transfection, Plasmid Preparation, Mutagenesis, Immunoprecipitation, Affinity Purification, Western Blot, Staining, Control, In Vivo, Ubiquitin Assay, Ubiquitin Proteomics, Expressing, Blocking Assay

Journal: PLoS ONE

Article Title: Investigation of FOXM1 as a Potential New Target for Melanoma

doi: 10.1371/journal.pone.0144241

Figure Lengend Snippet: PANC1 cells were used as a positive control. (a) The results of the quantitative RT-PCR analyses of FOXM1 mRNA expression. The relative transcript levels of FOXM1 normalized to the level in PANC1 are shown. (b) The expression of FOXM1 proteins in the malignant melanoma cell lines and NHEM. (c) The results of the quantitative RT-PCR analyses of the miR-370 mRNA expression. (d) The results of the semiquantitative RT-PCR using primers that can detect three splicing variants: FOXM1a (472bp), FOXM1b (323bp) and FOXM1c (368bp).

Article Snippet: Sections of paraffin-embedded melanomas and nevus tissue samples were stained with a monoclonal mouse anti-FOXM1 antibody (clone 3A9; Abnova, Taipei, Taiwan), monoclonal mouse anti-BRAFV600E antibody (clone VE1; Spring Bioscience, Pleasanton, CA) and monoclonal rabbit anti-phospho-AKT (Ser473) antibody (Cell Signaling Technologies, Tokyo, Japan).

Techniques: Positive Control, Quantitative RT-PCR, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Investigation of FOXM1 as a Potential New Target for Melanoma

doi: 10.1371/journal.pone.0144241

Figure Lengend Snippet: The FOXM1 expression (normalized to GAPDH) in the patients with primary melanoma (n = 25), metastatic melanoma (n = 9) and nevi (n = 10) is shown. The bars indicate the median values.

Article Snippet: Sections of paraffin-embedded melanomas and nevus tissue samples were stained with a monoclonal mouse anti-FOXM1 antibody (clone 3A9; Abnova, Taipei, Taiwan), monoclonal mouse anti-BRAFV600E antibody (clone VE1; Spring Bioscience, Pleasanton, CA) and monoclonal rabbit anti-phospho-AKT (Ser473) antibody (Cell Signaling Technologies, Tokyo, Japan).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Investigation of FOXM1 as a Potential New Target for Melanoma

doi: 10.1371/journal.pone.0144241

Figure Lengend Snippet: Representative images of the immunohistochemical staining for FOXM1 in primary malignant melanoma (a, b, c, d, e) and nevus tissues samples (f, g, h). Hematoxylin and eosin staining (a, f: × 40) and FOXM1 immunohistochemistry (b, g: × 40, c, h: × 400). Negative controls using an isotype monoclonal antibody were presented in d and e (d: × 40, e: × 400). Melanin granules are indicated by blue staining, although they did not exhibit FOXM1 expression. Bars: 500 μm (a, b, d, f, g), 50 μm (c, e, h).

Article Snippet: Sections of paraffin-embedded melanomas and nevus tissue samples were stained with a monoclonal mouse anti-FOXM1 antibody (clone 3A9; Abnova, Taipei, Taiwan), monoclonal mouse anti-BRAFV600E antibody (clone VE1; Spring Bioscience, Pleasanton, CA) and monoclonal rabbit anti-phospho-AKT (Ser473) antibody (Cell Signaling Technologies, Tokyo, Japan).

Techniques: Immunohistochemical staining, Staining, Immunohistochemistry, Expressing

Journal: PLoS ONE

Article Title: Investigation of FOXM1 as a Potential New Target for Melanoma

doi: 10.1371/journal.pone.0144241

Figure Lengend Snippet: The results of the immunohistochemical analysis of FOXM1.

Article Snippet: Sections of paraffin-embedded melanomas and nevus tissue samples were stained with a monoclonal mouse anti-FOXM1 antibody (clone 3A9; Abnova, Taipei, Taiwan), monoclonal mouse anti-BRAFV600E antibody (clone VE1; Spring Bioscience, Pleasanton, CA) and monoclonal rabbit anti-phospho-AKT (Ser473) antibody (Cell Signaling Technologies, Tokyo, Japan).

Techniques: Immunohistochemical staining

Journal: PLoS ONE

Article Title: Investigation of FOXM1 as a Potential New Target for Melanoma

doi: 10.1371/journal.pone.0144241

Figure Lengend Snippet: The results of the immunohistochemical analysis of FOXM1, BRAFV600E and p-AKT.

Article Snippet: Sections of paraffin-embedded melanomas and nevus tissue samples were stained with a monoclonal mouse anti-FOXM1 antibody (clone 3A9; Abnova, Taipei, Taiwan), monoclonal mouse anti-BRAFV600E antibody (clone VE1; Spring Bioscience, Pleasanton, CA) and monoclonal rabbit anti-phospho-AKT (Ser473) antibody (Cell Signaling Technologies, Tokyo, Japan).

Techniques: Immunohistochemical staining

Journal: PLoS ONE

Article Title: Investigation of FOXM1 as a Potential New Target for Melanoma

doi: 10.1371/journal.pone.0144241

Figure Lengend Snippet: The correlation between FOXM1 expression and the tumor thickness.

Article Snippet: Sections of paraffin-embedded melanomas and nevus tissue samples were stained with a monoclonal mouse anti-FOXM1 antibody (clone 3A9; Abnova, Taipei, Taiwan), monoclonal mouse anti-BRAFV600E antibody (clone VE1; Spring Bioscience, Pleasanton, CA) and monoclonal rabbit anti-phospho-AKT (Ser473) antibody (Cell Signaling Technologies, Tokyo, Japan).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Investigation of FOXM1 as a Potential New Target for Melanoma

doi: 10.1371/journal.pone.0144241

Figure Lengend Snippet: A comparison of the overall survival between the patients positive for FOXM1 and those negative for expression, as determined using immunohistochemical staining. The p -values were determined using the log-rank test.

Article Snippet: Sections of paraffin-embedded melanomas and nevus tissue samples were stained with a monoclonal mouse anti-FOXM1 antibody (clone 3A9; Abnova, Taipei, Taiwan), monoclonal mouse anti-BRAFV600E antibody (clone VE1; Spring Bioscience, Pleasanton, CA) and monoclonal rabbit anti-phospho-AKT (Ser473) antibody (Cell Signaling Technologies, Tokyo, Japan).

Techniques: Comparison, Expressing, Immunohistochemical staining, Staining

Journal: PLoS ONE

Article Title: Investigation of FOXM1 as a Potential New Target for Melanoma

doi: 10.1371/journal.pone.0144241

Figure Lengend Snippet: The human melanoma cell lines, MeWo and SK-MEL28, were transfected with control and FOXM1 siRNA. Twenty-four hours after treatment, the quantitative RT-PCR analyses were carried out (a,). Seventy-two hours after treatment, a Western blotting analysis and the BrdU cell proliferation assay were performed (b, c). The p -values were determined using the Mann–Whitney U-test. * p < 0.05.

Article Snippet: Sections of paraffin-embedded melanomas and nevus tissue samples were stained with a monoclonal mouse anti-FOXM1 antibody (clone 3A9; Abnova, Taipei, Taiwan), monoclonal mouse anti-BRAFV600E antibody (clone VE1; Spring Bioscience, Pleasanton, CA) and monoclonal rabbit anti-phospho-AKT (Ser473) antibody (Cell Signaling Technologies, Tokyo, Japan).

Techniques: Transfection, Control, Quantitative RT-PCR, Western Blot, BrdU Cell Proliferation Assay, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Investigation of FOXM1 as a Potential New Target for Melanoma

doi: 10.1371/journal.pone.0144241

Figure Lengend Snippet: The expression of FOXM1 was assessed using a Western blotting analysis. The human melanoma cell lines were treated with 10 μM of MEK1 siRNA for 72 hours.

Article Snippet: Sections of paraffin-embedded melanomas and nevus tissue samples were stained with a monoclonal mouse anti-FOXM1 antibody (clone 3A9; Abnova, Taipei, Taiwan), monoclonal mouse anti-BRAFV600E antibody (clone VE1; Spring Bioscience, Pleasanton, CA) and monoclonal rabbit anti-phospho-AKT (Ser473) antibody (Cell Signaling Technologies, Tokyo, Japan).

Techniques: Expressing, Western Blot

Journal: PLoS ONE

Article Title: Investigation of FOXM1 as a Potential New Target for Melanoma

doi: 10.1371/journal.pone.0144241

Figure Lengend Snippet: (a) The expression of activated AKT was assessed by a Western blotting analysis using phospho-specific anti-AKT antibodies. (b) The results of the Western blotting analysis of the cell lysates from the human melanoma cell lines treated with LY294002 (30 μM) and an AKT inhibitor (20 μM) for 24 hours. The levels of FOXM1, p-AKT (ser 473) and AKT were determined. (c) Melanoma cell lines were transfected with control or FOXM1 siRNA, and a Western blotting was carried out with p-AKT (ser 473), AKT and FOXM1 antibodies 72 hours after treatment.

Article Snippet: Sections of paraffin-embedded melanomas and nevus tissue samples were stained with a monoclonal mouse anti-FOXM1 antibody (clone 3A9; Abnova, Taipei, Taiwan), monoclonal mouse anti-BRAFV600E antibody (clone VE1; Spring Bioscience, Pleasanton, CA) and monoclonal rabbit anti-phospho-AKT (Ser473) antibody (Cell Signaling Technologies, Tokyo, Japan).

Techniques: Expressing, Western Blot, Transfection, Control