Journal: Journal of Ovarian Research

Article Title: NB compounds are potent and efficacious FOXM1 inhibitors in high-grade serous ovarian cancer cells

doi: 10.1186/s13048-024-01421-4

Figure Lengend Snippet: RT-qPCR primers

Article Snippet: Primary antibodies included anti- FOXM1 (Cell Signaling Technology, CST, Danvers, MA, USA) (CST #5436, 1:1,000), p-FOXM1 (Thr600) (CST #14655, 1:1,000–1:2,000), AURKB (Abcam, Waltham, MA, USA, #2254, 1:1,000), CCNB1 (CST #4138, 1:1,000–1:2,000), CDC25B (CST #9525, 1:1,000–1:2000), PLK1 (CST #4513, 1:1,000) FOXA1 (CST #53528, 1:1,000–1:2,000)), FOXK2 (CST #12008, 1:1,000), FOXO3a (CST #12829, 1:10,000), cleaved PARP (cl-PARP) (CST #5625, 1:1,000), ubiquitin (CST #14049, 1:20,000). alpha-tubulin (CST #2144, 1:5,000), β-actin (Santa Cruz Biotechnology, Dallas, TX, USA, # 47778, 1:10,000), and Lamin B1 (CST #12586, 1:1,000).

Techniques: Sequencing

Journal: Journal of Ovarian Research

Article Title: NB compounds are potent and efficacious FOXM1 inhibitors in high-grade serous ovarian cancer cells

doi: 10.1186/s13048-024-01421-4

Figure Lengend Snippet: Potency and efficacy of FOXM1 inhibitors in HGSOC cell lines a

Article Snippet: Primary antibodies included anti- FOXM1 (Cell Signaling Technology, CST, Danvers, MA, USA) (CST #5436, 1:1,000), p-FOXM1 (Thr600) (CST #14655, 1:1,000–1:2,000), AURKB (Abcam, Waltham, MA, USA, #2254, 1:1,000), CCNB1 (CST #4138, 1:1,000–1:2,000), CDC25B (CST #9525, 1:1,000–1:2000), PLK1 (CST #4513, 1:1,000) FOXA1 (CST #53528, 1:1,000–1:2,000)), FOXK2 (CST #12008, 1:1,000), FOXO3a (CST #12829, 1:10,000), cleaved PARP (cl-PARP) (CST #5625, 1:1,000), ubiquitin (CST #14049, 1:20,000). alpha-tubulin (CST #2144, 1:5,000), β-actin (Santa Cruz Biotechnology, Dallas, TX, USA, # 47778, 1:10,000), and Lamin B1 (CST #12586, 1:1,000).

Techniques:

Journal: Journal of Ovarian Research

Article Title: NB compounds are potent and efficacious FOXM1 inhibitors in high-grade serous ovarian cancer cells

doi: 10.1186/s13048-024-01421-4

Figure Lengend Snippet: Selectivity of FOXM1 inhibitors in HGSOC cells vs. FTE cells a

Article Snippet: Primary antibodies included anti- FOXM1 (Cell Signaling Technology, CST, Danvers, MA, USA) (CST #5436, 1:1,000), p-FOXM1 (Thr600) (CST #14655, 1:1,000–1:2,000), AURKB (Abcam, Waltham, MA, USA, #2254, 1:1,000), CCNB1 (CST #4138, 1:1,000–1:2,000), CDC25B (CST #9525, 1:1,000–1:2000), PLK1 (CST #4513, 1:1,000) FOXA1 (CST #53528, 1:1,000–1:2,000)), FOXK2 (CST #12008, 1:1,000), FOXO3a (CST #12829, 1:10,000), cleaved PARP (cl-PARP) (CST #5625, 1:1,000), ubiquitin (CST #14049, 1:20,000). alpha-tubulin (CST #2144, 1:5,000), β-actin (Santa Cruz Biotechnology, Dallas, TX, USA, # 47778, 1:10,000), and Lamin B1 (CST #12586, 1:1,000).

Techniques:

Journal: Journal of Ovarian Research

Article Title: NB compounds are potent and efficacious FOXM1 inhibitors in high-grade serous ovarian cancer cells

doi: 10.1186/s13048-024-01421-4

Figure Lengend Snippet: Protein expression and NB compound dose response in HGSOC and FTE cells. A Western blot analyses of FOXM1, FOXM1-P (P-Threonine 600), CCNB1, FOXA1, FOXK2, and FOXO3a expression in CAOV3 (HGSOC), OVCAR4 (HGSOC), and FT282-C11 (immortalized FTE cells). β-actin staining is a shown as a protein loading control. B-C Dose–response curves of CAOV3 (red), OVCAR4 (blue), and FT282-C11 (yellow) cells treated with B NB-73 or C NB-115 for 72 h. Cell viability was analyzed by CyQuant assay. Three independent trials were performed with three technical replicates per trial. Values represent mean ± SEM. Curves were created using nonlinear regression with least squares (ordinary) fit in GraphPad Prism. IC50 values are indicated below the graphs

Article Snippet: Primary antibodies included anti- FOXM1 (Cell Signaling Technology, CST, Danvers, MA, USA) (CST #5436, 1:1,000), p-FOXM1 (Thr600) (CST #14655, 1:1,000–1:2,000), AURKB (Abcam, Waltham, MA, USA, #2254, 1:1,000), CCNB1 (CST #4138, 1:1,000–1:2,000), CDC25B (CST #9525, 1:1,000–1:2000), PLK1 (CST #4513, 1:1,000) FOXA1 (CST #53528, 1:1,000–1:2,000)), FOXK2 (CST #12008, 1:1,000), FOXO3a (CST #12829, 1:10,000), cleaved PARP (cl-PARP) (CST #5625, 1:1,000), ubiquitin (CST #14049, 1:20,000). alpha-tubulin (CST #2144, 1:5,000), β-actin (Santa Cruz Biotechnology, Dallas, TX, USA, # 47778, 1:10,000), and Lamin B1 (CST #12586, 1:1,000).

Techniques: Expressing, Western Blot, Staining, Control, CyQUANT Assay

Journal: Journal of Ovarian Research

Article Title: NB compounds are potent and efficacious FOXM1 inhibitors in high-grade serous ovarian cancer cells

doi: 10.1186/s13048-024-01421-4

Figure Lengend Snippet: NB compound treatment suppresses FOXM1 and FOXM1-P expression in HGSOC cells. A Western blot analysis of FOXM1, FOXM1-P, and β-actin (loading control) expression in CAOV3 cells treated with the indicated concentrations of NB-73 or NB-115 for 48 h. Numerical values below the images indicate protein expression normalized by β-actin, and red numbers indicate reduced expression relative to DMSO control. B Quantified western blot data from panel A , with data from three biological replicates plotted. Two-way ANOVA with Dunnett’s multiple comparisons test, error bars indicate mean ± SD. C-D As in panels A - B , but in OVCAR4 cells treated for 72 h. E Western blot analysis of FOXM1, FOXM1-P, Lamin B1 (nuclear isolation control), α-tubulin (cytoplasm isolation control), and β-actin (overall loading control) expression in cytoplasmic and nuclear protein fractions from CAOV3 cells treated with the indicated concentrations of NB-73 and NB-115 for 48 h. Numerical values below the images indicate protein expression normalized to β-actin and red numbers indicate reduced expression relative to the DMSO control. F as in E , except in OVCAR4 cells treated for 72 h

Article Snippet: Primary antibodies included anti- FOXM1 (Cell Signaling Technology, CST, Danvers, MA, USA) (CST #5436, 1:1,000), p-FOXM1 (Thr600) (CST #14655, 1:1,000–1:2,000), AURKB (Abcam, Waltham, MA, USA, #2254, 1:1,000), CCNB1 (CST #4138, 1:1,000–1:2,000), CDC25B (CST #9525, 1:1,000–1:2000), PLK1 (CST #4513, 1:1,000) FOXA1 (CST #53528, 1:1,000–1:2,000)), FOXK2 (CST #12008, 1:1,000), FOXO3a (CST #12829, 1:10,000), cleaved PARP (cl-PARP) (CST #5625, 1:1,000), ubiquitin (CST #14049, 1:20,000). alpha-tubulin (CST #2144, 1:5,000), β-actin (Santa Cruz Biotechnology, Dallas, TX, USA, # 47778, 1:10,000), and Lamin B1 (CST #12586, 1:1,000).

Techniques: Expressing, Western Blot, Control, Isolation

Journal: Journal of Ovarian Research

Article Title: NB compounds are potent and efficacious FOXM1 inhibitors in high-grade serous ovarian cancer cells

doi: 10.1186/s13048-024-01421-4

Figure Lengend Snippet: NB compound treatment suppresses FOXM1 target protein and gene expression in HGSOC cells. A Western blot analysis of FOXM1 targets and β-actin expression in CAOV3 cells treated with the indicated concentrations of NB-73 or NB-115 for 48 h. Numerical values below the images indicate protein expression normalized by β-actin and red numbers indicate reduced expression relative to DMSO control. B Quantified western blot data from panel A , data from three biological replicates are plotted. Two-way ANOVA with Dunnett’s multiple comparisons test, error bars indicate mean ± SD. C-D As in panels A - B , but in OVCAR4 cells treated for 72 h. E RT-qPCR analysis of FOXM1 target gene expression in CAOV3 cells treated with the indicated concentration of NB-73 or NB-115 for 24 h. Line indicates median, two-way ANOVA with Tukey’s multiple comparison test. F As in panel E , except in OVCAR4 cells treated for 72 h

Article Snippet: Primary antibodies included anti- FOXM1 (Cell Signaling Technology, CST, Danvers, MA, USA) (CST #5436, 1:1,000), p-FOXM1 (Thr600) (CST #14655, 1:1,000–1:2,000), AURKB (Abcam, Waltham, MA, USA, #2254, 1:1,000), CCNB1 (CST #4138, 1:1,000–1:2,000), CDC25B (CST #9525, 1:1,000–1:2000), PLK1 (CST #4513, 1:1,000) FOXA1 (CST #53528, 1:1,000–1:2,000)), FOXK2 (CST #12008, 1:1,000), FOXO3a (CST #12829, 1:10,000), cleaved PARP (cl-PARP) (CST #5625, 1:1,000), ubiquitin (CST #14049, 1:20,000). alpha-tubulin (CST #2144, 1:5,000), β-actin (Santa Cruz Biotechnology, Dallas, TX, USA, # 47778, 1:10,000), and Lamin B1 (CST #12586, 1:1,000).

Techniques: Gene Expression, Western Blot, Expressing, Control, Quantitative RT-PCR, Targeted Gene Expression, Concentration Assay, Comparison

Journal: Journal of Ovarian Research

Article Title: NB compounds are potent and efficacious FOXM1 inhibitors in high-grade serous ovarian cancer cells

doi: 10.1186/s13048-024-01421-4

Figure Lengend Snippet: NB-73 treatment suppresses FOXM1 protein prior to FOXM1 mRNA in CAOV3 cells. A Western blot analysis of FOXM1, FOXM1-P, and β-actin expression in CAOV3 cells treated with 2.5 μM NB-73 for the indicated time points. B RT-qPCR of FOXM1 gene expression in CAOV3 cells treated with 2.5 μM NB-73 for the indicated time points. Error bars indicate mean ± SD, unpaired t-test. C FOXM1 protein and FOXM1 mRNA from three independent experiments as described in panels A and B , plotted together. Error bars indicate mean ± SD, two-way ANOVA with Dunnett’s multiple comparisons test

Article Snippet: Primary antibodies included anti- FOXM1 (Cell Signaling Technology, CST, Danvers, MA, USA) (CST #5436, 1:1,000), p-FOXM1 (Thr600) (CST #14655, 1:1,000–1:2,000), AURKB (Abcam, Waltham, MA, USA, #2254, 1:1,000), CCNB1 (CST #4138, 1:1,000–1:2,000), CDC25B (CST #9525, 1:1,000–1:2000), PLK1 (CST #4513, 1:1,000) FOXA1 (CST #53528, 1:1,000–1:2,000)), FOXK2 (CST #12008, 1:1,000), FOXO3a (CST #12829, 1:10,000), cleaved PARP (cl-PARP) (CST #5625, 1:1,000), ubiquitin (CST #14049, 1:20,000). alpha-tubulin (CST #2144, 1:5,000), β-actin (Santa Cruz Biotechnology, Dallas, TX, USA, # 47778, 1:10,000), and Lamin B1 (CST #12586, 1:1,000).

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Gene Expression

Journal: Journal of Ovarian Research

Article Title: NB compounds are potent and efficacious FOXM1 inhibitors in high-grade serous ovarian cancer cells

doi: 10.1186/s13048-024-01421-4

Figure Lengend Snippet: NB compound treatment mediated FOXM1 suppression in HGSOC cells is rescued by MG132 co-treatment. A Western blot analysis of FOXM1 and total ubiquitin expression in OVCAR4 cells treated with the indicated compounds for 24 h. Protein quantification is provided below each protein band, and protein expression is normalized to DMSO control (lane 1). Ponceau S staining shows total protein loading. Protein levels were quantified using Fiji software . B As in panel A , except in COV318 cells treated for 24 h

Article Snippet: Primary antibodies included anti- FOXM1 (Cell Signaling Technology, CST, Danvers, MA, USA) (CST #5436, 1:1,000), p-FOXM1 (Thr600) (CST #14655, 1:1,000–1:2,000), AURKB (Abcam, Waltham, MA, USA, #2254, 1:1,000), CCNB1 (CST #4138, 1:1,000–1:2,000), CDC25B (CST #9525, 1:1,000–1:2000), PLK1 (CST #4513, 1:1,000) FOXA1 (CST #53528, 1:1,000–1:2,000)), FOXK2 (CST #12008, 1:1,000), FOXO3a (CST #12829, 1:10,000), cleaved PARP (cl-PARP) (CST #5625, 1:1,000), ubiquitin (CST #14049, 1:20,000). alpha-tubulin (CST #2144, 1:5,000), β-actin (Santa Cruz Biotechnology, Dallas, TX, USA, # 47778, 1:10,000), and Lamin B1 (CST #12586, 1:1,000).

Techniques: Western Blot, Ubiquitin Proteomics, Expressing, Control, Staining, Software

Journal: Journal of Ovarian Research

Article Title: NB compounds are potent and efficacious FOXM1 inhibitors in high-grade serous ovarian cancer cells

doi: 10.1186/s13048-024-01421-4

Figure Lengend Snippet: NB compound treatment mediated FOXM1 suppression in CAOV3 cells after drug washout. A Experimental schematic, created with BioRender. B-D Western blot analysis of FOXM1, FOXM1-P, CCNB1, and β-actin expression in CAOV3 cells treated as indicated in panel A , for B 24 h, C 48 h, and D 72 h post-treatment. E CyQuant cell viability assay data for CAOV3 cells treated with NB-73. The three curves compare data from no washout (72 h), 6 h drug exposure then washout (6 h), and 3 h drug exposure then washout (3 h). IC50 values for each condition are indicated in brackets. Error bars indicate mean ± SD, non-linear regression curve. F As in E , except for NB-115 treatment

Article Snippet: Primary antibodies included anti- FOXM1 (Cell Signaling Technology, CST, Danvers, MA, USA) (CST #5436, 1:1,000), p-FOXM1 (Thr600) (CST #14655, 1:1,000–1:2,000), AURKB (Abcam, Waltham, MA, USA, #2254, 1:1,000), CCNB1 (CST #4138, 1:1,000–1:2,000), CDC25B (CST #9525, 1:1,000–1:2000), PLK1 (CST #4513, 1:1,000) FOXA1 (CST #53528, 1:1,000–1:2,000)), FOXK2 (CST #12008, 1:1,000), FOXO3a (CST #12829, 1:10,000), cleaved PARP (cl-PARP) (CST #5625, 1:1,000), ubiquitin (CST #14049, 1:20,000). alpha-tubulin (CST #2144, 1:5,000), β-actin (Santa Cruz Biotechnology, Dallas, TX, USA, # 47778, 1:10,000), and Lamin B1 (CST #12586, 1:1,000).

Techniques: Western Blot, Expressing, CyQUANT Assay, Viability Assay

Journal: Journal of Ovarian Research

Article Title: NB compounds are potent and efficacious FOXM1 inhibitors in high-grade serous ovarian cancer cells

doi: 10.1186/s13048-024-01421-4

Figure Lengend Snippet: NB compound treatment mediated FOXM1 suppression is independent of apoptosis. A Western blot analysis of FOXM1, FOXM1-P, cl-PARP, and β-actin expression in CAOV3 cells treated with the indicated compounds for 48 h. Numerical values below the images indicate protein expression normalized by β-actin and red numbers indicate reduced expression relative to DMSO control. B Quantified western blot data from panel A ; data from three biological replicates are plotted. Error bars indicate mean ± SD. C Western blot analysis of CCNB1, PLK1, AURKB, and CDC25B expression in CAOV3 cells treated with the indicated compounds for 48 h. D Quantified western blot data from panel C ; data from three biological replicates are plotted. Error bars indicate mean ± SD

Article Snippet: Primary antibodies included anti- FOXM1 (Cell Signaling Technology, CST, Danvers, MA, USA) (CST #5436, 1:1,000), p-FOXM1 (Thr600) (CST #14655, 1:1,000–1:2,000), AURKB (Abcam, Waltham, MA, USA, #2254, 1:1,000), CCNB1 (CST #4138, 1:1,000–1:2,000), CDC25B (CST #9525, 1:1,000–1:2000), PLK1 (CST #4513, 1:1,000) FOXA1 (CST #53528, 1:1,000–1:2,000)), FOXK2 (CST #12008, 1:1,000), FOXO3a (CST #12829, 1:10,000), cleaved PARP (cl-PARP) (CST #5625, 1:1,000), ubiquitin (CST #14049, 1:20,000). alpha-tubulin (CST #2144, 1:5,000), β-actin (Santa Cruz Biotechnology, Dallas, TX, USA, # 47778, 1:10,000), and Lamin B1 (CST #12586, 1:1,000).

Techniques: Western Blot, Expressing, Control

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