Fig 2: 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.

Fig 3: Up-regulation of FOXM1 transcription by E6 is through increased NKX2-1 binding to the FOXM1 promoter. (A) Schematic diagram of FOXM1 promoter–driven luciferase reporters: FOXM1 (- 1436/+ 1)-Luc, FOXM1 (- 766/+ 1)-Luc, and FOXM1 (- 1436/-766)-Luc. NKX2-1-A, NKX2-1-B, and NKX2-1-C possess putative NKX2-1 binding sites. (B) The three promoter constructs were co-transfected into SiHa, C33A, GNM, OECM-1, TL-1, and A549 cells for luciferase reporter assay after 48 hours. Luciferase activity was measured at 48-hour post-transfection. In all experiments, the relative luciferase activity was shown as the fold activation relative to that of the control cells. (C) SiHa, GNM, and TL-1 cells were transfected with various doses of E6-knockdown plasmid as indicated. C33A, OECM-1, and A549 cells were transfected with various doses of E6-overexpressing plasmid. The total amount of transfected DNA was kept constant by adding a control vector. The luciferase activity was measured at 48-hour post-transfection. In all experiments, the relative luciferase activity was shown as the fold activation relative to that of the control cells. SiHa, GNM, and TL-1 cells were transfected with various doses of E6-knockdown plasmid as indicated. C33A, OECM-1, and A549 cells were transfected with various doses of E6-overexpressing plasmid. In addition, SiHa, GNM, and TL-1 cells were co-transfected with NKX2-1-overexpressing (5 µg) and E6-knockdown (5 µg) plasmids. C33A, OECM-1, and A549 cells were co-transfected with shNKX2-1-knockdown (5 µg) and E6-overexpressing (5 µg) plasmids. These lysates were immunoprecipitated with anti–NKX2-1-conjugated beads. The immunoprecipitates were analyzed through SDS-PAGE, followed by immunoblot analysis with anti–NKX2-1 antibody. The input control was 30% of the cell extract without any treatment. The binding activity of NKX2-1 onto the FOXM1 promoter was evaluated through chromatin immunoprecipitation. The chromatin was isolated and immunoprecipitated with an antibody specific for NKX2-1.

Fig 4: E6-induced FOXM1 expression promotes cell invasiveness and stemness through activating ß-catenin/TCF signaling pathway in OCSCC cells. (A) Western blot analysis of the total levels of E6, p53, FOXM1, cyclin D1, c-Myc, Nanog, Oct4, and ß-catenin; cytoplasmic and nuclear levels of ß-catenin and FOXM1 in GNM and OECM-1 cells that were transfected with a vector (control, NC, or VC), FOXM1, shFOXM1, E6, or shE6 plasmids, respectively. ß-Actin, a-tubulin, and SP1 were used as the protein loading controls of whole-cell extracts, as well as cytoplasmic and nuclear proteins. TOPFlash and FOPFlash activity levels were determined through luciferase reporter activity assay. FOPFlash was used as a negative control. ß-Gal served as an internal control. Values are given as means ± SEMs for triplicate samples. (B) Western blot analysis of the total levels of E6, p53, FOXM1, cyclin D1, c-Myc, Nanog, Oct4, and ß-catenin in GNM cells that were, respectively, transfected with a vector (control, NC, or VC), shE6, FOXM1, and shß-catenin plasmids. The sphere formation and invasive cells were photographed (bottom) and quantified (upper right) The scale bar represents a length of 50 µm. (D) Western blot analysis of the total levels of E6, p53, FOXM1, cyclin D1, c-Myc, Nanog, Oct4, and ß-catenin in OECM-1–E6 cells on treatment with thiostrepton at various dosages. Quantitative real-time PCR (right) was used to analyze the levels of FOXM1, Nanog, Oct4, and c-Myc mRNA expression. The data shown are the means ± SDs of three independent experiments.

Fig 5: Knockdown of FoxM1 expression in HT-29 and HCT-116 cells reduces their CD44+ subpopulations and their sensitivity to thiostrepton. Vector-control and FoxM1-knockdown clones were established from HT-29 (29 V, 29sh1 and 29sh2) and HCT-116 (116 V, 116sh1, and 116sh2) cells by retroviral infection. (a) The protein levels of FoxM1 and CD44 in the aforementioned clones were analyzed by western blotting. Actin signal served as a loading control. (b) The percentage of CD44+ subpopulations (numbers in the top right quadrant) in 29 V and 29sh1 clones and 116 V and 116sh1 cells were measured by flow cytometry. (c) The IC50 values of thiostrepton on the aforementioned four clones were determined as described in the ‘Materials and Methods'. *P<0.05 when compared with their respective vector control clones by Student's t-test