Journal: Nature Communications

Article Title: Nuclear AURKA acquires kinase-independent transactivating function to enhance breast cancer stem cell phenotype

doi: 10.1038/ncomms10180

Figure Lengend Snippet: ( a ) DBD/AURKA-DBD, DBD-reporter and pRL-TK were co-transfected into 293T for 24 h. Immunoblotting (IB; left panel) and dual-luciferase reporter (right panel) analysis were performed. ( b ) DBD/AURKA-DBD, DBD-reporter and pRL-TK were co-transfected into 293T cells. After 12 h, cells were treated with VX-680, MLN8237 or Aurora-A inhibitor I for 12 h. IB (left panel) and dual-luciferase reporter assay (right panel) were performed. ( c ) Diagram showing potential TADs in AURKA. ( d ) DBD/AURKA-DBD, DBD-reporter and pRL-TK were co-transfected into 293T cells for 24 h. IB (left panel) and dual-luciferase reporter assay (right panel) were performed. ( e ) STRING analysis showing connections among AURKA-regulated genes. ( f ) ER/AER, MYC promoter/basic reporter and pRL-TK were co-transfected into MDA-MB-231 cells. After 6 h, AURKA nuclear translocation was induced by treating cells with 200 nM OHT for 18 h. Dual-luciferase reporter assay was performed. ( g ) Treatment was the same as in f . Expression of indicated genes was determined via real-time PCR and IB analysis. ( h ) Treatment was similar to d , except for using MYC promoter reporter instead of DBD reporter. IB (left panel) and a dual-luciferase reporter (right panel) analysis were performed. ( i ) AURKA or empty vector was co-transfected into MDA-MB-231 cells with truncated MYC promoter (MYC) or basic reporter (Vec) along with pRL-TK. After 24 h, dual-luciferase reporter assays were performed. ( j ) MDA-MB-231 cells were subjected for ChIP analysis of MYC promoter occupancy. Results were normalized with the input. ( k ) VX-680-treated MDA-MB-231 cells (48 h) were subjected for ChIP analysis of MYC promoter occupancy. ( l ) Cells were cultured in suspended or adherent condition with same medium for 7 days. S1 nuclease protection assay (SNPA) was performed to examine MYC P1 and P2 transcripts. Control gene, β-2-microglobulin (B2M) , mRNA in parallel identical samples were also determined. ( m ) BCSC and non-BCSC populations were isolated according to CD24 expression. SNPA was performed. ( n ) ER/AER plasmids were transfected into 293T cells. After 6 h, AURKA nuclear translocation was induced by treating cells with 200 nM OHT for 18 h. SNPA was performed. ( o ) DBD/AURKA-DBD were transfected into 293T cells for 24 h. SNPA was performed. Bars represent the means±s.e.m. of three independent experiments (analysis of variance (ANOVA) followed by least significant difference (LSD) test; * P <0.05, ** P <0.01, *** P <0.001).

Article Snippet: Enhanced green fluorescent protein-fused full-length and truncated AURKA were purchased from GenePharma.

Techniques: Transfection, Western Blot, Luciferase, Reporter Assay, Translocation Assay, Expressing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Cell Culture, Control, Isolation

Journal: Nature Communications

Article Title: Nuclear AURKA acquires kinase-independent transactivating function to enhance breast cancer stem cell phenotype

doi: 10.1038/ncomms10180

Figure Lengend Snippet: ( a ) AURKA-interacting proteins were identified using SILAC assay (red area). MYC promoter-regulating proteins were previously reported (blue area). Proteins presented in both categories were selected for further analysis. ( b ) Thirty-five combinations of the amino acid derived from AURKA and hnRNP K with high probabilities of interactions were used to compile a dot plot. The amino acids in orange box were located in the nucleotide 283–333 region of the AURKA sequence. ( c ) The simulated interaction diagram of AURKA and hnRNP K. KI domain was shown. ( d ) Nuclear/cytoplasmic protein fractions of MDA-MB-231 cells were subjected to IP and immunoblotting (IB) using antibodies as indicated. ( e ) hnRNP K-ECFP- and AURKA-EYFP-co-transfected 293T cells were subjected to FRET efficiency analysis. ROI1 and ROI2 were selected for the analysis in the cytoplasmic and nuclear regions, respectively. Enhanced cyan fluorescent protein (ECFP)- and enhanced yellow fluorescent protein (EYFP)-co-transfected cells were used as negative controls. Scale bar, 50 μm. ( f ) Twenty micrograms of WT or NLS deletion mutant hnRNP K were co-transfected with Flag-AURKA–enhanced green fluorescent protein (EGFP) into 293T cells for 24 h. Cells were then subjected to IP and IB using antibodies as indicated. ( g ) Twenty micrograms of WT or NLS deletion mutant hnRNP K were co-transfected with Flag-AURKA–EGFP in 293T for 24 h. Cytoplasmic and nuclear proteins were separated and subjected to IP and IB using antibodies as indicated. ( h ) Twenty micrograms of AER was co-transfected with Flag-tagged hnRNP K into 293T cells. After 6 h, AURKA nuclear translocation was induced by treatment with 200 nM OHT for 18 h. Cytoplasmic and nuclear proteins were then separated and subjected to IP and IB using antibodies as indicated. ( i ) Samples 1–3 were subjected to IHC staining of AURKA. Scale bar, 100 μm. ( j ) The lysates of samples 1–3 were subjected to IB using antibodies as indicated. ( k ) The lysates of samples 1–3 were subjected to IP and IB using antibodies as indicated. Bars represent the means±s.e.m. of three independent experiments (analysis of variance (ANOVA) followed by least significant difference (LSD) test; * P <0.05, ** P <0.01, *** P <0.001).

Article Snippet: Enhanced green fluorescent protein-fused full-length and truncated AURKA were purchased from GenePharma.

Techniques: Multiplex sample analysis, Derivative Assay, Sequencing, Western Blot, Transfection, Mutagenesis, Translocation Assay, Immunohistochemistry

Journal: Nature Communications

Article Title: Nuclear AURKA acquires kinase-independent transactivating function to enhance breast cancer stem cell phenotype

doi: 10.1038/ncomms10180

Figure Lengend Snippet: ( a ) Enhanced green fluorescent protein (EGFP)-fused truncated forms of AURKA were transfected into 293T cells for 24 h. The localization of EGFP-fused truncated forms of AURKA were analysed via confocal microscopy. Scale bar, 25 μm. ( b ) DsRed-fused 333–383 region of AURKA was transfected into 293T cells for 24 h. The localization of DsRed-fused 333–383 region of AURKA was analysed by confocal microscopy. Scale bar, 25 μm. ( c , d ) MDA-MB-231 cells were co-infected with lentivirus expressing AURKA shRNA (shAURKA/sh Control) and the truncated protein (1–383 or 1–333). Puromycin (1 μg ml −1 ) and blasticidin (5 μg ml −1 ) selected cells were subjected to immunoblotting (IB) analysis ( c ) or CD24 low /CD44 high population analysis via flow cytometry ( d ). ( e ) The cells in c were subjected to mammospheres culture assay for 6 days. The left panel shows the distribution pattern according to mammosphere size (Kruskal–Wallis test followed by Dunn's multiple comparison test, *** P <0.001). The right panel showed the number of Φ>60 μm mammospheres. ( f ) AURKA (1–333) was fused with ER via a nuclear localization sequence to generate ER-NLS-AUR333 fusion protein (upper panel). MDA-MB-231 cells were co-infected with lentivirus expressing AURKA shRNA (shAURKA/sh Control) and the fusion protein (ER-NLS-AUR333). The cells were then cultured in fresh medium for 48 h in the presence or absence of 200 nM OHT. Puromycin (1 μg ml −1 ) and blasticidin (5 μg ml −1 ) selected cells were harvested for CD24/CD44 staining and analysis via flow cytometry (lower panel). ( g ) The cells in f were cultured in mammosphere medium with or without 200 nM OHT. The cells were then photographed and quantified at the end of the first-round mammosphere culture (6 days) and the secondary passaging (additional 6 days). The left panel shows the distribution pattern of mammosphere size from MDA-MB-231 (Kruskal–Wallis test followed by Dunn's multiple comparison test, *** P <0.001). The right panel shows the number of mammosphere (Φ>60 μm). Data were presented as the means±s.e.m. of three independent experiments (analysis of variance (ANOVA) followed by least significant difference (LSD) test; * P <0.05; ** P <0.01; *** P <0.001).

Article Snippet: Enhanced green fluorescent protein-fused full-length and truncated AURKA were purchased from GenePharma.

Techniques: Transfection, Confocal Microscopy, Infection, Expressing, shRNA, Control, Western Blot, Flow Cytometry, Comparison, Sequencing, Cell Culture, Staining, Passaging