Journal: Hepatology (Baltimore, Md.)

Article Title: Reciprocal Regulation Between Forkhead Box M1/NF-κB and Methionine Adenosyltransferase 1A Drives Liver Cancer.

doi: 10.1002/hep.31196

Figure Lengend Snippet: Figure 1. FOXM1 and MAT1A mRNA levels in HCC and CCA, and their influences on survival curves in HCC. A) Relative FOXM1 and MMP-7 mRNA levels from seven patients with CCA compared to normal liver tissues. *p < 0.05 vs. normal human liver tissues. B) Relative MAT1A and FOXM1 mRNA levels from 143 patients with HCC (TU) compared to adjacent non- tumorous tissues (AD). *p < 0.05 vs. AD. C) Pearson correlation analysis of MAT1A and FOXM1 mRNA levels in 143 HCC specimens and in 52 HCCs with vascular invasion. D) Pearson correlation analysis of FOXM1 and MAT1A mRNA levels in HCC Samples (N=366) from the Cancer Genome Atlas (TCGA) dataset. E and F) Kaplan-Meier survival

Article Snippet: Migration and invasion assays Migration and invasion assays were performed as described. (29) MATα1 and FOXM1 interaction Recombinant human MATα1 (CAT#: enz-493-b) was purchased from PROSPEC (East Brunswick NJ) and FOXM1 (CAT#: TP76157 was from OriGENE (Rockville, MD).

Techniques:

Journal: Hepatology (Baltimore, Md.)

Article Title: Reciprocal Regulation Between Forkhead Box M1/NF-κB and Methionine Adenosyltransferase 1A Drives Liver Cancer.

doi: 10.1002/hep.31196

Figure Lengend Snippet: Figure 2. Protein expression of MATα1, FOXM1, p50, and p65 from HCC and CCA, adjacent and normal liver tissues. A) Representative immunohistochemistry (IHC) staining of MATα1, FOXM1, p50, and p65 from normal liver, non-tumorous tissue adjacent to CCA (CCA-AD), CCA (CCA-TU), HCC-AD, and HCC-TU. All are at 200X magnification. Higher magnification (400X) of boxed areas is shown below for each IHC. B) MATα1, FOXM1, p50, and p65 protein levels from three pairs of CCA and adjacent tissues (CCA-AD), and three normal liver specimens on western blotting. Numbers below the blots are densitometric values, expressed as percent of normal liver. *p < 0.05. vs. normal liver; #p < 0.05 vs. adjacent tissues. C) MATα1, FOXM1, p50, and p65 protein levels in four pairs of HCC and adjacent tissues (HCC-AD), and four normal liver specimens on western blotting. *p < 0.05 vs. normal liver tissues; #p < 0.05 vs. adjacent tissues.

Article Snippet: Migration and invasion assays Migration and invasion assays were performed as described. (29) MATα1 and FOXM1 interaction Recombinant human MATα1 (CAT#: enz-493-b) was purchased from PROSPEC (East Brunswick NJ) and FOXM1 (CAT#: TP76157 was from OriGENE (Rockville, MD).

Techniques: Expressing, Immunohistochemistry, Western Blot

Journal: Hepatology (Baltimore, Md.)

Article Title: Reciprocal Regulation Between Forkhead Box M1/NF-κB and Methionine Adenosyltransferase 1A Drives Liver Cancer.

doi: 10.1002/hep.31196

Figure Lengend Snippet: Figure 3. Reciprocal regulation between FOXM1/NF-κB and MAT1A. A) Protein levels of MATα1, FOXM1, p50, p65, HNF4, AFP, CYP2E1, and ACTIN were measured using western blotting in primary mouse hepatocytes at the time of isolation (0 hour = 0h) and up to 6 hours (6h) after plating. B) Protein levels of MATα1, FOXM1, p50 and p65 after overexpressing wild type MAT1A (1A OV), catalytic mutant of MAT1A (1Am) or siRNA knockdown (1A si) as compared to empty vector (EV) or scramble (SC) controls, respectively, in HepG2 cells. Numbers below the blots are densitometric values, expressed as percent of EV or SC in mean ± SEM from three experiments. *p < 0.05 vs. EV or SC; #p<0.05 vs. 1Am. C-D) Protein levels of MATα1, FOXM1, p50, and p65 after FOXM1 expression (FOXM1 OV) or siRNA knockdown (FOXM1 si) in HepG2 (C) and SAMe-D cells, which were also co-transfected with EV or MAT1A (D). Numbers below the blots are densitometric values, expressed as percent of EV or SC. Results are expressed as mean percent of control ± SEM from three experiments. *p < 0.05 vs. respective controls. E)

Article Snippet: Migration and invasion assays Migration and invasion assays were performed as described. (29) MATα1 and FOXM1 interaction Recombinant human MATα1 (CAT#: enz-493-b) was purchased from PROSPEC (East Brunswick NJ) and FOXM1 (CAT#: TP76157 was from OriGENE (Rockville, MD).

Techniques: Western Blot, Isolation, Mutagenesis, Knockdown, Plasmid Preparation, Expressing, Transfection, Control

Journal: Hepatology (Baltimore, Md.)

Article Title: Reciprocal Regulation Between Forkhead Box M1/NF-κB and Methionine Adenosyltransferase 1A Drives Liver Cancer.

doi: 10.1002/hep.31196

Figure Lengend Snippet: Figure 4. Interaction between FOXM1 and MATα1. A) In vitro pull-down assay using immobilized recombinant MATα1 or FOXM1 and Immunoblot (IB) for MATα1 and FOXM1. B) Western blotting and co-immunoprecipitation (Co-IP) were used to detect MATα1 and FOXM1 protein levels and their interactions in human HCC and adjacent tissues (AD). C) MATα1 and FOXM1 protein levels and their interactions in mouse CCA and control livers.

Article Snippet: Migration and invasion assays Migration and invasion assays were performed as described. (29) MATα1 and FOXM1 interaction Recombinant human MATα1 (CAT#: enz-493-b) was purchased from PROSPEC (East Brunswick NJ) and FOXM1 (CAT#: TP76157 was from OriGENE (Rockville, MD).

Techniques: In Vitro, Pull Down Assay, Recombinant, Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay, Control

Journal: Hepatology (Baltimore, Md.)

Article Title: Reciprocal Regulation Between Forkhead Box M1/NF-κB and Methionine Adenosyltransferase 1A Drives Liver Cancer.

doi: 10.1002/hep.31196

Figure Lengend Snippet: Figure 5. FOXM1 and NF-κB regulate MAT1A expression through FOX binding sites. A) EMSA was done using labeled probes containing three FOX binding motifs of the MAT1A promoter as shown above and 100ng of recombinant FOXM1, MATα1, p50, p65, PHB1 alone or combined (left panel), nuclear proteins (0.2μg) from HCC, CCA, and respective adjacent non-tumorous tissues (AD) (middle panel), and with supershift using antibodies to MATα1, p50, FOXM1, and p65 (right panel). Results represent three or more independent experiments. Probe and IgG only served as negative controls. B) MAT1A promoter activity was measured in HepG2 cells following transient transfection with serial deletion constructs. Cells were treated during the last 24 hours of the transfection with DMSO or FDI-6 (5μM). Results represent mean ± SEM from four experiments done in triplicates, *p < 0.05 vs. DMSO. C) FOX binding sites and their mutants in the human MAT1A promoter were created as described in Materials and Methods. D). Effect of T0 (5μM) on the wild type MAT1A promoter (−839/30, 1A WT) or MAT1A promoter mutated

Article Snippet: Migration and invasion assays Migration and invasion assays were performed as described. (29) MATα1 and FOXM1 interaction Recombinant human MATα1 (CAT#: enz-493-b) was purchased from PROSPEC (East Brunswick NJ) and FOXM1 (CAT#: TP76157 was from OriGENE (Rockville, MD).

Techniques: Expressing, Binding Assay, Labeling, Recombinant, Activity Assay, Transfection, Construct

Journal: Hepatology (Baltimore, Md.)

Article Title: Reciprocal Regulation Between Forkhead Box M1/NF-κB and Methionine Adenosyltransferase 1A Drives Liver Cancer.

doi: 10.1002/hep.31196

Figure Lengend Snippet: Figure 6. Effects of MAT1A and p65 on the FOXM1 promoter. A) HepG2 cells were treated with MAT1A OV or si, or FOXM1 OV or si, and respective controls for 24 hours. ChIP analysis with anti-FOXM1 or MATα1 antibody, and then re- ChIP with anti-MATα1, p65, p50 antibodies after FOXM1 ChIP were performed as described in Materials and Methods. Representative results from three experiments are shown. B) Activities of the wild-type (WT) and FOX binding site mutants (MU) of the FOXM1 promoter after MAT1A OV and si treatment for 24 hours in HepG2 cell. Results are expressed as mean % of EV or SC ± SEM from three experiments done in duplicates *p < 0.05 vs. EV or. SC. C) Promoter activities of the WT and FOX binding site MU of the FOXM1 promoter after p65 OV for 24 hours in the HepG2 cell. Results are expressed as mean % of EV ± SEM from three experiments done in duplicates. *p < 0.05 vs. EV. Results represent at least three independent experiments done in duplicate.

Article Snippet: Migration and invasion assays Migration and invasion assays were performed as described. (29) MATα1 and FOXM1 interaction Recombinant human MATα1 (CAT#: enz-493-b) was purchased from PROSPEC (East Brunswick NJ) and FOXM1 (CAT#: TP76157 was from OriGENE (Rockville, MD).

Techniques: Binding Assay

Journal: Hepatology (Baltimore, Md.)

Article Title: Reciprocal Regulation Between Forkhead Box M1/NF-κB and Methionine Adenosyltransferase 1A Drives Liver Cancer.

doi: 10.1002/hep.31196

Figure Lengend Snippet: Figure 7. Effects of MAT1A and FOXM1 on cell proliferation, migration and invasion. A) Effects of varying MAT1A and FOXM1 expressions on HepG2 cell migration. Quantitative values are summarized in the graph to the right. Results are expressed as mean % of respective controls ± SEM from three experiments done in duplicates. *p < 0.05 vs. EV +EV or SC+SC; #p < 0.05 vs. FOXM1 OV or siRNA; †p < 0.05 vs. MAT1A OV or siRNA. B) Dose-response effect of FDI-6 treatment on the MTT assay in HepG2 cells. Results are expressed as mean % of DMSO ± SEM from three experiments done in duplicates. *p < 0.05 vs. DMSO; #p < 0.05 vs FDI-6 (10uM). C) Time-dependent effect of FDI-6 (5uM) treatment on the MTT assay in OKER and SAMe-D cells. Results are expressed as mean % of DMSO ± SEM from three experiments done in duplicates. *p < 0.05 vs. DMSO. D) Representative invasion images of OKER and SAMe-D cells after FDI-6 (5uM) treatment. Quantitative analysis is summarized in the graph to the right. Results are expressed as mean % of DMSO ± SEM from three experiments done in duplicates. *p < 0.05 vs. DMSO. E)

Article Snippet: Migration and invasion assays Migration and invasion assays were performed as described. (29) MATα1 and FOXM1 interaction Recombinant human MATα1 (CAT#: enz-493-b) was purchased from PROSPEC (East Brunswick NJ) and FOXM1 (CAT#: TP76157 was from OriGENE (Rockville, MD).

Techniques: Migration, MTT Assay

Journal: Hepatology (Baltimore, Md.)

Article Title: Reciprocal Regulation Between Forkhead Box M1/NF-κB and Methionine Adenosyltransferase 1A Drives Liver Cancer.

doi: 10.1002/hep.31196

Figure Lengend Snippet: Figure 8. Effects of T0 and FDI-6 on expression of MATα1, FOXM1, p50 and p65, and tumor growth. A) MATα1, FOXM1, p50, and p65 levels after T0 treatment (5μM) in HepG2 cells. Results are expressed as mean % of DMSO control ± SEM from three experiments done in duplicates. *p<0.05 vs. DMSO. B) Representative pictures of liver xenograft tumors at day 28 after injection of HepG2 cells in DMSO (left) and T0 treatment (right) groups. Results are expressed as mean % of DMSO from n=8 per group. C) FOXM1 and MAT1A mRNA levels from T0 (25 mg/kg/d) or DMSO treated xenograft tumor tissues. Results are expressed as mean % of control ± SEM from eight mice/group. D) Representative H&E and IHC pictures are shown from n = 8 each. Original magnification for H&E and IHCs is X200. E) Protein expression of MATα1, FOXM1, p50, and p65 after FDI-6 treatment in HepG2 cells. Results are expressed as mean % of control ± SEM from three experiments done in triplicates. *p < 0.05 vs. DMSO. F) Representative pictures of liver syngeneic tumors at day 11 after injection of OKER cells in DMSO (left) and FDI-6 treatment (right, 25 mg/kg/d

Article Snippet: Migration and invasion assays Migration and invasion assays were performed as described. (29) MATα1 and FOXM1 interaction Recombinant human MATα1 (CAT#: enz-493-b) was purchased from PROSPEC (East Brunswick NJ) and FOXM1 (CAT#: TP76157 was from OriGENE (Rockville, MD).

Techniques: Expressing, Control, Injection

Journal: Molecular Oncology

Article Title: DRP1 contributes to head and neck cancer progression and induces glycolysis through modulated FOXM1/MMP12 axis

doi: 10.1002/1878-0261.13212

Figure Lengend Snippet: FOXM1 regulates the expression and transcription activity of MMP12 in HNC cells. (A) Potential FOXM1 binding sites in human MMP12 promoter region. (B,C) QRT‐PCR, western blotting and luciferase assays indicating the expression and luciferase activity of MMP12 in FOXM1‐depleted HNC cells. Quantification of relative FOXM1 and MMP12 expressions are shown. (D) ChIP assays were performed to confirm the binding of FOXM1 to the MMP12 promoter in SAS and HSC‐3 cells using an anti‐FOXM1 antibody. Isotype IgGs were used as a negative control. (E) The protein expression level of MMP12 was investigated in FOXM1‐depleted cells transfected with Flag‐MMP12 by western blotting. Quantification of relative Flag‐MMP12 expression is shown. (F,G) The growth and motility of siFOXM1 with MMP12 overexpression in HNC transfectants was determined. All data are presented as mean ± SD of three independent experiments. Significance was calculated using t ‐test. In (F), statistical analyses were performed using one‐way ANOVA followed by Tukey’s multiple comparison’s test. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Consistently, similar results were observed in forced expression of FOXM1 in SAS cells treated with Mdivi‐1 (Fig. ).

Techniques: Expressing, Activity Assay, Binding Assay, Quantitative RT-PCR, Western Blot, Luciferase, Negative Control, Transfection, Over Expression

Journal: Molecular Oncology

Article Title: DRP1 contributes to head and neck cancer progression and induces glycolysis through modulated FOXM1/MMP12 axis

doi: 10.1002/1878-0261.13212

Figure Lengend Snippet: MMP12 expression is essential for DRP1/FOXM1 regulation in HNC cells. (A) The mRNA and protein expression levels of FOXM1 in siDRP1 cells were examined. Quantification of relative FOXM1 expression is shown. (B,C) Western blotting, QPCR and luciferase activity analysis of MMP12 were determined in SAS cells transfected with FOXM1 or vector control in combination with siDRP1 or negative control. Quantification of relative DRP1, Flag‐FOXM1 and MMP12 expressions is shown. (D,E) Western blotting, QPCR and luciferase activity of MMP12 were analyzed in SAS cells transfected with FOXM1 or vector control in combination with Mdivi‐1 treatment. Quantification of relative DRP1, Flag‐FOXM1, and MMP12 expressions is shown. (F) IHC staining patterns of the HNC tumor tissues for DRP1, FOXM1 and MMP12. Scale bar: 100 µm. All data presented as mean ± SD of three independent experiments. Significance calculated using t ‐test. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Consistently, similar results were observed in forced expression of FOXM1 in SAS cells treated with Mdivi‐1 (Fig. ).

Techniques: Expressing, Western Blot, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Control, Negative Control, Immunohistochemistry

Journal: Molecular Oncology

Article Title: DRP1 contributes to head and neck cancer progression and induces glycolysis through modulated FOXM1/MMP12 axis

doi: 10.1002/1878-0261.13212

Figure Lengend Snippet: The correlation among DRP1, FOXM1and MMP12 protein expression in HNC tumor tissues.

Article Snippet: Consistently, similar results were observed in forced expression of FOXM1 in SAS cells treated with Mdivi‐1 (Fig. ).

Techniques: Expressing

Journal: International Journal of Biological Sciences

Article Title: Mechanistic Insights into the FOXM1/BUB1 axis-Mediated Oncogenic Signaling in Hepatocellular Carcinoma

doi: 10.7150/ijbs.125454

Figure Lengend Snippet: FOXM1 promotes the proliferation of HCC cells in vitro . (A) The expression of FOXM1 in tumor tissues and adjacent tissues of patients with HCC analyzed by IHC. (B) The expression of Ki-67 in tumor tissues of patients with HCC analyzed by IHC. (C) The differential expression of FOXM1 between tumor tissues and adjacent tissues of patients with HCC analyzed by IHC. (D) Correlation analysis of FOXM1 and Ki-67 IHC scores in tumor tissues of patients with HCC. (E) Box plots for the differential expression of FOXM1 between LIHC tissues and adjacent tissues in TCGA. (F) ROC curves for the relationship between FOXM1 expression and the prognosis of HCC patients. (G) Correlation between FOXM1 expression levels and survival of HCC patients. (H) Distribution of FOXM1 mRNA expression in HCC cell lines. (I) Expression of FOXM1 in HUH7, HepG2, and SMMC-7721 cells analyzed by Western blot. (J) Effect of FOXM1 shRNA and FOXM1 cDNA on the viability of HUH7 cells. (K) Effects of FOXM1 on the formation of HUH7 colonies. (L) Effects of FOXM1 on the apoptosis of HUH7 cells. (M) Effects of FDI-6 on the proliferation of HUH7 cells. (N) Effects of FDI-6 on the formation of HUH7 colonies. (O) Effects of FOXM1 shRNA and FOXM1 cDNA on mouse weight. (P) Effects of FOXM1 shRNA and FOXM1 cDNA on tumor volume. (Q) The photos of tumor nodules in each group. (R) Effects of FOXM1 shRNA and FOXM1 cDNA on tumor weight. (S) Effects of FOXM1 shRNA and FOXM1 cDNA on tumor proliferation. (T) The expression of Ki-67 and FOXM1 in xenograft tumor analyzed by IHC. Images were randomly selected from five replicates. Data from three independent experiments were analyzed by one-way ANOVA: * P<0.05, ** P<0.01 vs. control group; # P<0.05, ## P<0.01 vs. FOXM1 KD group.

Article Snippet: The differential expression of FOXM1 in HCC tissues and adjacent normal tissues from the human protein atlas was analyzed using the online tool ( https://www.proteinatlas.org/ ).

Techniques: In Vitro, Expressing, Quantitative Proteomics, Western Blot, shRNA, Control

Journal: International Journal of Biological Sciences

Article Title: Mechanistic Insights into the FOXM1/BUB1 axis-Mediated Oncogenic Signaling in Hepatocellular Carcinoma

doi: 10.7150/ijbs.125454

Figure Lengend Snippet: FOXM1-regulated genes involve in DNA repair, cell cycle, stemness and EMT. (A) GO function and KEGG pathway enrichment analysis of DEGs between FOXM1 KD and NC KD in HUH7 cells. (B) . GO function and KEGG pathway enrichment analysis of top 50 targets in the PPI network of FOXM1. (C) . Volcano plot for the DEGs involved in DNA repair and cell cycle analyzed by RNA-seq. (D) Volcano plot for the DEGs involved in cell stemness, invasion and migration. (E) . The relationship between FOXM1 expression and HCC cell stemness in TCGA. (F) . Differential expression of genes between LIHC tissues and adjacent tissues in TCGA. (G) . ROC curves for the relationship between gene expression and HCC prognosis. Data were statistically analyzed using one-way ANOVA: * P<0.05 compared with adjacent tissues.

Article Snippet: The differential expression of FOXM1 in HCC tissues and adjacent normal tissues from the human protein atlas was analyzed using the online tool ( https://www.proteinatlas.org/ ).

Techniques: RNA Sequencing, Migration, Expressing, Quantitative Proteomics, Gene Expression

Journal: International Journal of Biological Sciences

Article Title: Mechanistic Insights into the FOXM1/BUB1 axis-Mediated Oncogenic Signaling in Hepatocellular Carcinoma

doi: 10.7150/ijbs.125454

Figure Lengend Snippet: FOXM1 promotes DNA repair and G2/M progression on HCC cells. (A) Effects of FOXM1 on DNA damage in HUH7 and HepG2 cells analyzed by comet assay. (B) Effects of FOXM1 on γH2AX expression analyzed by IF. (C) Q-PCR analysis of the effects of FOXM1 on the expression of DNA repair-related genes. (D) Analysis of the correlation between FOXM1 and DNA repair-related gene expressions in LIHC. (E) Effects of FOXM1 on cell cycle progression in HUH7 cells. (F) percentage of cell cycle at G0/G1, S, and G2/M phases. (G) Q-PCR analysis of the effects of FOXM1 on the expression of genes regulating G2/M transition. (H) Analysis of the correlation between FOXM1 and cell cycle related gene expressions in HCC. (I) Effects of FDI-6 on DNA damage in HUH7 cells. (J) Effects of FDI-6 on the expression of DNA-repair related genes in HUH7 cells. (K) Effects of FDI-6 on cell cycle progression in HUH7 cells. (L) Effects of FDI-6 on cell cycle-related genes in HUH7 cells. Images were randomly selected from five replicates. Data from three independent experiments were statistically analyzed using one-way ANOVA: * P < 0.05, ** P < 0.01 vs. NC KD group; # P < 0.05, ## P < 0.01 vs. FOXM1 KD group.

Article Snippet: The differential expression of FOXM1 in HCC tissues and adjacent normal tissues from the human protein atlas was analyzed using the online tool ( https://www.proteinatlas.org/ ).

Techniques: Single Cell Gel Electrophoresis, Expressing

Journal: International Journal of Biological Sciences

Article Title: Mechanistic Insights into the FOXM1/BUB1 axis-Mediated Oncogenic Signaling in Hepatocellular Carcinoma

doi: 10.7150/ijbs.125454

Figure Lengend Snippet: FOXM1 drives cell stemness, invasion, and migration in HCC cells. (A) Effects of FOXM1 on CD44 expression analyzed by IF in HUH7 cells. (B) Effects of FOXM1 on CD44 expression in HepG2 cells. (C) Effects of FOXM1 on the expression of stemness-related genes in HUH7 cells. (D) Effects of FDI-6 on CD44 expression in HUH7 cells. (E) Effects of FDI-6 on the expression of stemness-related genes in HUH7 cells. (F) Effects of FOXM1 shRNA and FDI-6 on the formation of HUH7 three-dimensional spheres. (G) Analysis of the correlation between FOXM1 and cell cycle-related gene expressions in HCC. (H) Effects of FOXM1 on the invasion and migration of HUH7 and HepG2 cells. (I) Q-PCR analysis of the effects of FOXM1 on EMT-related gene expression in HUH7 cells. (J) Effects of FDI-6 on the invasion and migration of HUH7 cells. (K) Q-PCR analysis of the effects of FDI-6 on EMT-related gene expression in HUH7 cells. (L) Analysis of the correlation between FOXM1 and EMT-related gene expressions in HCC. Images were randomly selected from five replicates. Data from three independent experiments were statistically analyzed using one-way ANOVA: * P < 0.05, ** P < 0.01 vs. NC KD group; # P < 0.05, ## P < 0.01 vs. FOXM1 KD group.

Article Snippet: The differential expression of FOXM1 in HCC tissues and adjacent normal tissues from the human protein atlas was analyzed using the online tool ( https://www.proteinatlas.org/ ).

Techniques: Migration, Expressing, shRNA, Gene Expression

Journal: International Journal of Biological Sciences

Article Title: Mechanistic Insights into the FOXM1/BUB1 axis-Mediated Oncogenic Signaling in Hepatocellular Carcinoma

doi: 10.7150/ijbs.125454

Figure Lengend Snippet: FOXM1 promotes BUB1 expression at transcriptional level. (A) Venn diagram of overlapping genes identified by RNA-seq and FOXM1 PPI network. (B) Analysis the association between key gene expression and HCC patient survival. (C) The expression of BUB1 between HCC tissues and adjacent tissues from clinical patients analyzed by IHC. (D) IHC scores of BUB1 between HCC tissues and adjacent tissues from clinical patients. (E) Correlation analysis of BUB1 and Ki-67 IHC scores in tumor tissues of patients with HCC. (F) Correlation analysis of BUB1 and FOXM1 IHC scores in tumor tissues of patients with HCC. (G) Co-IP analysis of the interaction between FOXM1 and BUB1 in HUH7 cells. (H) Effects of FOXM1 shRNA and BUB1 shRNA on the expression of FOXM1 and BUB1 analyzed by Q-PCR. (I) The expression of FOXM1 and BUB1 analyzed by Western blot. (J) ChIP-qPCR analysis of the binding of FOXM1 and BUB1 promoter in HUH7 cells. (K) The sequences at the -293 bp of the BUB1-WT promoter and the BUB1-Mut promoter. (L) The binding of FOXM1 and BUB1 promoter at the -293 bp sequence GTAAACC analyzed by dual luciferase reporter assay. (M) Q-PCR analysis of BUB1 expression in different HCC cells. (N) Western blot analysis of BUB1 expression in different HCC cells. (O) KEGG pathway enrichment analysis of top 50 targets in the PPI network of BUB1. (P) Venn diagram of overlapping genes identified by RNA-seq, FOXM1 PPI network, and BUB1 PPI network. (Q) correlation analysis of BUB1 expression and LIHC cell stemness in TCGA. (R) Effect of BUB1 on the inhibitory role of FOXM1 shRNA in the proliferation of HUH7 cells. (S) Effects of BUB1 on the inhibition of FOXM1 shRNA in HUH7 colony formation. (T) Effects of BUB1 on the promotion of FOXM1 shRNA in HUH7 cell apoptosis. (U) Effects of BUB1 shRNA on HUH7 xenograft tumor volumes. (V) Photos of HUH7 xenograft tumors in each group. (W) Effects of BUB1 shRNA on HUH7 xenograft tumor weight. (X) Effects of BUB1 shRNA on Ki-67 and BUB1 expression in each group. Images were randomly selected from five replicates. Data from three independent experiments were analyzed by one-way ANOVA: * P<0.05, ** P<0.01 vs. NC KD group, # P < 0.05, ## P < 0.01 vs. FOXM1 KD group.

Article Snippet: The differential expression of FOXM1 in HCC tissues and adjacent normal tissues from the human protein atlas was analyzed using the online tool ( https://www.proteinatlas.org/ ).

Techniques: Expressing, RNA Sequencing, Gene Expression, Co-Immunoprecipitation Assay, shRNA, Western Blot, ChIP-qPCR, Binding Assay, Sequencing, Luciferase, Reporter Assay, Inhibition

Journal: International Journal of Biological Sciences

Article Title: Mechanistic Insights into the FOXM1/BUB1 axis-Mediated Oncogenic Signaling in Hepatocellular Carcinoma

doi: 10.7150/ijbs.125454

Figure Lengend Snippet: Knockdown of BUB1 enhances HCC cell sensitivity to FOXM1 inhibitor FDI-6. (A) Effect of BUB1 shRNA on FDI-6-mediated inhibition of colony formation in HUH7 cells. (B) Effect of BUB1 shRNA on FDI-6-induced apoptosis. (C) Effect of BUB1 shRNA on FDI-6-induced DNA damage. (D) Effect of BUB1 shRNA on the G2/M phase arrest caused by FDI-6. (E) Western blot analysis of BUB1 shRNA and FDI-6 effects on DNA repair-related gene expression. (F) Western blot analysis of BUB1 shRNA and FDI-6 effects on cell cycle-related gene expression. (G) IF analysis of BUB1 shRNA and FDI-6 effects on CD44 expression. (H) Western blot analysis of BUB1 shRNA and FDI-6 effects on stemness-related gene expression. (I) Effects of BUB1 shRNA on FDI-6 mediated suppression of HCC cell invasion and migration. (J) Western blot analysis of BUB1 shRNA and FDI-6 effects on EMT-related gene expression. (K) Effects of BUB1 shRNA and FDI-6 on mouse weight. (L) Effects of BUB1 shRNA and FDI-6 on tumor volume. (M) The photos of HUH7 xenograft tumors in each group. (N) Effects of BUB1 shRNA and FDI-6 on tumor weight. (O) The inhibition ratios of BUB1 shRNA and FDI-6 on tumor volume and weight. (P) IHC analysis of the effects of BUB1 shRNA and FDI-6 on Ki-67 and FOXM1 expression Images were randomly selected from five replicates. Data from three independent experiments were statistically analyzed using one-way ANOVA: * P<0.05, ** P<0.01 vs. NC KD group, # P < 0.05, ## P < 0.01 vs. BUB1 KD+FDI-6 group.

Article Snippet: The differential expression of FOXM1 in HCC tissues and adjacent normal tissues from the human protein atlas was analyzed using the online tool ( https://www.proteinatlas.org/ ).

Techniques: Knockdown, shRNA, Inhibition, Western Blot, Gene Expression, Expressing, Migration

Journal: International Journal of Biological Sciences

Article Title: Mechanistic Insights into the FOXM1/BUB1 axis-Mediated Oncogenic Signaling in Hepatocellular Carcinoma

doi: 10.7150/ijbs.125454

Figure Lengend Snippet: FOXM1 inhibitors and BAY synergistically inhibit proliferation of HCC cells and tumors. (A) Effect of BAY and FDI-6 on the proliferation of HUH7 cells. (B) The CI values of the combinations of BAY and FDI-6 at different concentration ratios. (C) Effect of BAY, FDI-6 and their combination on the colony formation of HUH7 cells. (D) Effect of BAY, FDI-6 and their combination on the apoptosis of HUH7 cells. (E) The CI values of the combinations of TST and FDI-6 at different concentration ratios. (F) Effect of TST, FDI-6 and their combination on the colony formation of HUH7 cells. (G) The CI values of the combinations of RCM-1 and FDI-6 at different concentration ratios. (H) Effect of RCM-1, FDI-6 and their combination on the colony formation of HUH7 cells. (I) Effect of BAY, FDI-6 and their sequential combination on tumor volume. (J) Effect of BAY, FDI-6 and their sequential combination on tumor weight. (K) The photos of HUH7 xenograft tumors in each group. (L) Acute toxicity analysis of 100 mg/kg FDI-6, 100 mg/kg BAY, and their combination in mice. (M) Effect of BAY, FDI-6 and their sequential combination on mouse weight. (N) Effect of BAY, FDI-6 and their sequential combination on the heart, liver, spleen, lung and kidney in mice. (O) Effect of BAY, FDI-6 and their sequential combination on the expression of Ki-67, FOXM1, and BUB1 in HUH7 xenograft tumors. Images were randomly selected from five replicates. Data from three independent experiments were statistically analyzed using one-way ANOVA: * P<0.05, ** P<0.01 vs. control group, # P < 0.05, ## P < 0.01 vs. BAY-FDI-6 group.

Article Snippet: The differential expression of FOXM1 in HCC tissues and adjacent normal tissues from the human protein atlas was analyzed using the online tool ( https://www.proteinatlas.org/ ).

Techniques: Concentration Assay, Expressing, Control

Journal: International Journal of Biological Sciences

Article Title: Mechanistic Insights into the FOXM1/BUB1 axis-Mediated Oncogenic Signaling in Hepatocellular Carcinoma

doi: 10.7150/ijbs.125454

Figure Lengend Snippet: FOXM1/BUB1 axis drives HCC cell DNA repair, G2/M transition, stemness, migration, and invasion. (A) Effect of BAY, FDI-6 and their combination on DNA damage in HUH7 cells. (B) Effect of BAY, FDI-6 and their combination on DNA repair-related genes in HUH7 cells analyzed by Q-PCR. (C) Effect of BAY, FDI-6 and their combination on cell cycle progression in HUH7 cells. (D) Effect of BAY, FDI-6 and their combination on cell cycle-related genes in HUH7 cells analyzed by Q-PCR. (E) Effect of BAY, FDI-6 and their combination on CD44 expression in HUH7 cells analyzed by IF. (F) Q-PCR analysis of the effects of BAY, FDI-6 and their combination on the expression of cell cycle-related genes in HUH7 cells. (G) Effect of BAY, FDI-6 and their combination on the formation of HUH7 spheres. (H) Effect of BAY, FDI-6 and their combination on the migration and invasion of HUH7 cells. (I) Q-PCR analysis of the effects of BAY, FDI-6 and their combination on the expression of EMT-related genes in HUH7 cells. (J) The molecular mechanism of the FOXM1/BUB1 axis in regulating HCC malignancy. Images were randomly selected from five replicates. Data from three independent experiments were statistically analyzed using one-way ANOVA: * P<0.05, ** P<0.01 vs. control group, # P < 0.05, ## P < 0.01 vs. BAY-FDI-6 group.

Article Snippet: The differential expression of FOXM1 in HCC tissues and adjacent normal tissues from the human protein atlas was analyzed using the online tool ( https://www.proteinatlas.org/ ).

Techniques: Migration, Expressing, Control