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: Lentiviral recombination vectors encoding human FOXM1 complete DNA (FOXM1 cDNA) (pGV341-cFOXM1), human BUB1 complete DNA (BUB1 cDNA) (pGV341-cBUB1), empty vector control (pGV341-cNC), lentiviral shRNA vectors targeting FOXM1 (pGV112-shFOXM1), BUB1 (pGV112-shBUB1), and scrambled control (pGV112-shNC) were constructed and purchased from Genechem Co. Ltd. (Shanghai, China).

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: Inhibition of BUB1 suppresses HCC cell DNA repair, stemness, invasion, and migration. (A) Effects of BUB1 on DNA damage analyzed by comet assay. (B) Effects of BUB1 on γH2AX expression analyzed by IF. (C) Effects of BUB1 on the expression of DNA repair-related genes analyzed by Q-PCR. (D) Effects of BUB1 on cell cycle progression analyzed by flow cytometry. (E) Percentage of cell cycle at G0/G1, S, and G2/M phases. (F) Effects of BUB1 on the expression of cell cycle-related genes analyzed by Q-PCR. (G) Effects of BUB1 on CD44 expression in HUH7 cells analyzed by IF. (H) Effects of BUB1 on stemness-related gene expression in HUH7 cells analyzed by Q-PCR. (I) Effects of BUB1 on HUH7 cell invasion and migration. (J) Effects of BUB1 on HUH7 cell invasion and migration. 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 group.

Article Snippet: Lentiviral recombination vectors encoding human FOXM1 complete DNA (FOXM1 cDNA) (pGV341-cFOXM1), human BUB1 complete DNA (BUB1 cDNA) (pGV341-cBUB1), empty vector control (pGV341-cNC), lentiviral shRNA vectors targeting FOXM1 (pGV112-shFOXM1), BUB1 (pGV112-shBUB1), and scrambled control (pGV112-shNC) were constructed and purchased from Genechem Co. Ltd. (Shanghai, China).

Techniques: Inhibition, Migration, Single Cell Gel Electrophoresis, Expressing, Flow Cytometry, 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: 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: Lentiviral recombination vectors encoding human FOXM1 complete DNA (FOXM1 cDNA) (pGV341-cFOXM1), human BUB1 complete DNA (BUB1 cDNA) (pGV341-cBUB1), empty vector control (pGV341-cNC), lentiviral shRNA vectors targeting FOXM1 (pGV112-shFOXM1), BUB1 (pGV112-shBUB1), and scrambled control (pGV112-shNC) were constructed and purchased from Genechem Co. Ltd. (Shanghai, China).

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: Lentiviral recombination vectors encoding human FOXM1 complete DNA (FOXM1 cDNA) (pGV341-cFOXM1), human BUB1 complete DNA (BUB1 cDNA) (pGV341-cBUB1), empty vector control (pGV341-cNC), lentiviral shRNA vectors targeting FOXM1 (pGV112-shFOXM1), BUB1 (pGV112-shBUB1), and scrambled control (pGV112-shNC) were constructed and purchased from Genechem Co. Ltd. (Shanghai, China).

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: Lentiviral recombination vectors encoding human FOXM1 complete DNA (FOXM1 cDNA) (pGV341-cFOXM1), human BUB1 complete DNA (BUB1 cDNA) (pGV341-cBUB1), empty vector control (pGV341-cNC), lentiviral shRNA vectors targeting FOXM1 (pGV112-shFOXM1), BUB1 (pGV112-shBUB1), and scrambled control (pGV112-shNC) were constructed and purchased from Genechem Co. Ltd. (Shanghai, China).

Techniques: Migration, Expressing, Control