Journal: Oncogene

Article Title: Paclitaxel targets FOXM1 to regulate KIF20A in mitotic catastrophe and breast cancer paclitaxel resistance

doi: 10.1038/onc.2015.152

Figure Lengend Snippet: Paclitaxel-resistant MCF-7 cells exhibit upregulated expression levels of FOXM1 and KIF20A. ( a ) Protein expression levels of FOXM1, KIF20A, cyclin B1 and PARP in MCF-7 and MCF-7 Tax R cell lines were examined by western blotting after paclitaxel treatment at different time points indicated (left panel). Notably, there was no cleavage of PARP. qRT-PCR analysis determining the relative mRNA expression levels of FOXM1 and KIF20A in MCF-7 and MCF-7 Tax R cell lines after paclitaxel treatment. The bar graph represents an average of three independent experiments±s.d. ( n =3). ( b ) MCF-7 and MCF-7 Tax R cells transfected with Smart pool siRNA against FOXM1 or with non-silencing controls (NSC) were treated with 10 n M paclitaxel and harvested for FOXM1 and KIF20A expression analysis. FOXM1 and KIF20A mRNA levels were determined by qRT-PCR. Value is mean ±s.d. ( n =3). ( c ) The expression levels of FOXM1, KIF20A and β-tubulin were analysed by western blot analysis. ( d ) The expression levels of FOXM1, KIF20A and β-tubulin were analysed by western blot analysis in FoxM1 −/− and WT MEFs. ( e ) The expression levels of FOXM1, KIF20A and β-tubulin were also analysed by western blot analysis in MCF-7 cells transfected with control vector and the pmCherry-FOXM1 expression vector.

Article Snippet: MCF-7 cells were co-transfected with the human KIF20A luciferase reporter (WT or MUT), transfection control Renilla (pRL-TK; Promega, Southampton, UK) and pcDNA3-FOXM1 plasmids using FuGENE6 (Roche).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Transfection, Control, Plasmid Preparation

Journal: Oncogene

Article Title: Paclitaxel targets FOXM1 to regulate KIF20A in mitotic catastrophe and breast cancer paclitaxel resistance

doi: 10.1038/onc.2015.152

Figure Lengend Snippet: FOXM1 binds to the KIF20A promoter region in MCF-7 breast cancer cell line. ( a ) Schematic representation of the 5' region of the human KIF20A gene depicting the FHRE, the putative promoter regions cloned, the WT and the mutant (mut) promoter sequences, the ENCODE ChIP-seq data and the ChIP-qPCR amplicon. ( b ) The binding of FOXM1 to the human KIF20A promoter was examined by ChIP analysis in MCF-7 cells transfected with control vector or a FOXM1 expression vector (upper panel). The occupancy of the FHRE by FOXM1 was also examined in MCF-7 with and without 10μM thiostrepton treatment. ( c ) MCF-7 cells were transiently co-transfected with WT or mut KIF20A reporter plasmids and Renilla luciferase plasmid along with or without FOXM1 expression plasmids (10 μg). Twenty-four hours after transfection, cells were lysed, and the luciferase activity examined. Firefly luminescence signal was normalized with the Renilla luminescence signal. Value is mean±s.d. ( n =3). Statistical significance was determined by Student's t -test, two-sided (very significant ** P ⩽0.01).

Article Snippet: MCF-7 cells were co-transfected with the human KIF20A luciferase reporter (WT or MUT), transfection control Renilla (pRL-TK; Promega, Southampton, UK) and pcDNA3-FOXM1 plasmids using FuGENE6 (Roche).

Techniques: Clone Assay, Mutagenesis, ChIP-sequencing, ChIP-qPCR, Amplification, Binding Assay, Transfection, Control, Plasmid Preparation, Expressing, Luciferase, Activity Assay

Journal: Oncogene

Article Title: Paclitaxel targets FOXM1 to regulate KIF20A in mitotic catastrophe and breast cancer paclitaxel resistance

doi: 10.1038/onc.2015.152

Figure Lengend Snippet: Characterization of mitotic spindle defects in MCF-7 cells following FOXM1 or KIF20A depletion. MCF-7 cells were transfected with non-silencing controls (NSC), FOXM1 or KIF20A siRNA. Twenty-four hours after transfection, cells cultured on chamber slides were either untreated or treated with 5 n M paclitaxel for 24 h. Cells were then fixed, permeabilized and immunostained with antibody against α-tubulin (Green) and γ-tubulin (Red). Nuclei were counterstained with 4',6-diamidino-2-phenylindole (Blue). Mitotic cells were visualized with Leica TCS SP5 ( × 63 magnification). For each condition, images of at least 50 mitotic cells were captured. Representative confocal images are shown. The number of mitotic cells classified into either normal bipolar, bipolar with chromosome misalignment, monopolar or multipolar spindles was quantified. Results represent average of three independent experiments±s.d. Statistical significance was determined by Student's t -test, two-sided (not significant, ns; very significant ** P ⩽0.01).

Article Snippet: MCF-7 cells were co-transfected with the human KIF20A luciferase reporter (WT or MUT), transfection control Renilla (pRL-TK; Promega, Southampton, UK) and pcDNA3-FOXM1 plasmids using FuGENE6 (Roche).

Techniques: Transfection, Cell Culture

Journal: Oncogene

Article Title: Paclitaxel targets FOXM1 to regulate KIF20A in mitotic catastrophe and breast cancer paclitaxel resistance

doi: 10.1038/onc.2015.152

Figure Lengend Snippet: Depletion of KIF20A or FOXM1 inhibits cell proliferation and induces senescence in MCF-7 cells. ( a ) MCF-7 cells were transfected with either non-silencing control (NSC) siRNA, siRNA targeting FOXM1 or KIF20A. Twenty-four hours after transfection, 2000 cells were seeded in six-well plates, treated with 0, 1 or 3 n M of paclitaxel, grown for 15 days, and then stained with crystal violet (left panel). The result (right panel) represents an average of three independent experiments±s.d. Statistical significance was determined by Student's t -test, two-sided (* P ⩽0.05, ** P ⩽0.01, *** P ⩽0.005; n.s., non-significant). In parallel, ( b ) MCF-7 transfected with NSC, FOXM1 or KIF20A siRNA were seeded in six-well plates, treated with 0, 1 or 3 n M of paclitaxel. Five days after treatment, cells were stained for SAβ-gal activity. The graph shows the percentage of SAβ-gal-positive cells as measured from five different fields from three independent experiments. Bars represent mean±s.d. Statistical significance was determined by Student's t -test, two-sided (* P ⩽0.05, ** P ⩽0.01, *** P ⩽0.005, significant; n.s., non-significant).

Article Snippet: MCF-7 cells were co-transfected with the human KIF20A luciferase reporter (WT or MUT), transfection control Renilla (pRL-TK; Promega, Southampton, UK) and pcDNA3-FOXM1 plasmids using FuGENE6 (Roche).

Techniques: Transfection, Control, Staining, Activity Assay

Journal: Oncogene

Article Title: Paclitaxel targets FOXM1 to regulate KIF20A in mitotic catastrophe and breast cancer paclitaxel resistance

doi: 10.1038/onc.2015.152

Figure Lengend Snippet: Knockdown of FOXM1 or KIF20A suppresses cell proliferation and induces senescence in MCF-7 Tax R cells. ( a ) MCF-7 Tax R were transfected with either non-silencing control (NSC) siRNA, siRNA targeting FOXM1 or KIF20A. Twenty-four hours after transfection, 2000 cells were seeded in six-well plates, treated with 0, 1 or 3 n M of paclitaxel, grown for 15 days and then stained with crystal violet (left panel). The result (right panel) represents an average of three independent experiments±s.d. Statistical significance was determined by Student's t -test, two-sided (* P ⩽0.05, ** P ⩽0.01, significant; n.s., non-significant). In parallel, ( b ) MCF-7 Tax R transfected with NSC, FOXM1 or KIF20A siRNA were seeded in six-well plates, treated with 0, 1 or 3 n M of paclitaxel. Five days after treatment, cells were stained for SAβ-gal activity. The graph shows the percentage of SAβ-gal-positive cells as measured from five different fields from three independent experiments. Bars represent average±s.d. Statistical significance was determined by Student's t -test, two-sided (* P ⩽0.05, ** P ⩽0.01, *** P ⩽0.005, significant; n.s., non-significant).

Article Snippet: MCF-7 cells were co-transfected with the human KIF20A luciferase reporter (WT or MUT), transfection control Renilla (pRL-TK; Promega, Southampton, UK) and pcDNA3-FOXM1 plasmids using FuGENE6 (Roche).

Techniques: Knockdown, Transfection, Control, Staining, Activity Assay

Journal: Oncogene

Article Title: Paclitaxel targets FOXM1 to regulate KIF20A in mitotic catastrophe and breast cancer paclitaxel resistance

doi: 10.1038/onc.2015.152

Figure Lengend Snippet: ( a ) FOXM1 and KIF20A expression was assessed by immunohistochemistry using tissue-microarray constructed from 116 breast cancer patient samples. KIF20A was expressed in both cytoplasm and nucleus. Representative staining images of one patient with high FOXM1 and KIF20A expression and one with low expression are shown. Positive correlation between FOXM1 and KIF20A was observed. ( b ) KIF20A staining were detected in both nuclear and cytoplasmic compartments and were correlated with FOXM1 staining. Statistical analysis revealed that all three KIF20A scores (total, cytoplasmic and nuclear) were significantly correlated with FOXM1 expression. ( P =0.006, P =0.019 and P =0.034, respectively). P ⩽0.05, significant; P ⩽0.01, very significant; P ⩽0.005, very very significant.

Article Snippet: MCF-7 cells were co-transfected with the human KIF20A luciferase reporter (WT or MUT), transfection control Renilla (pRL-TK; Promega, Southampton, UK) and pcDNA3-FOXM1 plasmids using FuGENE6 (Roche).

Techniques: Expressing, Immunohistochemistry, Microarray, Construct, Staining

Journal: Oncogene

Article Title: Paclitaxel targets FOXM1 to regulate KIF20A in mitotic catastrophe and breast cancer paclitaxel resistance

doi: 10.1038/onc.2015.152

Figure Lengend Snippet: KIF20A overexpression significantly associated with poorer survival in breast cancer patients. ( a ) Kaplan–Meier survival analysis (SPSS) of all patients showed that nuclear KIF20A overexpression significantly associated with poorer survival ( n =100). ( b ) Kaplan–Meier survival analysis of patients received chemotherapy showed that nuclear KIF20A overexpression significantly associated with poorer survival ( n =60). ( c ) Kaplan–Meier survival analysis of patients FOXM1 and KIF20A mRNA overexpression significantly associated with poorer survival ( n =3455). P ⩽0.05, significant; P ⩽0.01, very significant; P ⩽0.005, very very significant.

Article Snippet: MCF-7 cells were co-transfected with the human KIF20A luciferase reporter (WT or MUT), transfection control Renilla (pRL-TK; Promega, Southampton, UK) and pcDNA3-FOXM1 plasmids using FuGENE6 (Roche).

Techniques: Over Expression