Journal: Molecular Cancer Research

Article Title: RNA-seq Reveals Aurora Kinase–Driven mTOR Pathway Activation in Patients with Sarcomatoid Metastatic Renal Cell Carcinoma

doi: 10.1158/1541-7786.mcr-14-0352

Figure Lengend Snippet: Figure 4. AURKA overexpression drives mTOR activation in Renca cells. Renca cell lines were transfected with an AURKA expression vector and 2 independent cell lines were selected. After several weeks of selection, lysates were harvested from both AURKA cell lines and parental Renca cells, separated by SDS-PAGE, and immunoblotting was performed for the indicated proteins (A), or RNA was harvested for RT-qPCR (B). Overexpression of AURKA caused increased expression of mTOR and S6K, as well as phosphorylation of both proteins. The increased expression of mTOR was not reflected at the transcript level, although the AURKA target ccnb1 was increased. Bars represent SE. ANOVA methods were used to derive P values (, P < 0.05).

Article Snippet: Cells were transfected with an AURKA expression vector (AddGene plasmid 8510; ref. 11) using Lipofectamine transfection reagents (Life Technologies), and 2 independent clones were selected using puromycin.

Techniques: Over Expression, Activation Assay, Transfection, Expressing, Plasmid Preparation, Selection, SDS Page, Western Blot, Quantitative RT-PCR, Phospho-proteomics

Journal: Human Cell

Article Title: Role of aurora kinase B in regulating resistance to paclitaxel in breast cancer cells

doi: 10.1007/s13577-022-00675-8

Figure Lengend Snippet: Correlation between AURKB expression level and resistance to PTX in breast cancer. a , b Measurement of cell viability by CCK-8 in breast cancer cells (MDA-MB-231 and BT549) and corresponding PTX-resistant cells (MDA-MB-231/PTX and BT549/PTX) that were treated with PTX at different concentrations for 48 h. c The relative contents of AURKB and phosphorylated AURKB in PTX-resistant cell line MDA-MB-231/PTX and wild-type cell line MDA-MB-231 as well as after Hesperadin (50 μM) treatment by Western blot. d The relative contents of AURKB and phosphorylated AURKB in PTX-resistant cell line BT549/PTX and wild-type cell line BT549 as well as after Hesperadin treatment by Western blot. e , f The cell survival rates of blank group, DMSO group and Hesperadin (200 nm) treatment group by CCK-8 at different time points after MDA-MB-231/PTX and BT549/PTX were treated by PTX (0.5 μM). Comparisons among groups were determined by one-way ANOVA with Tukey post hoc analysis. * P < 0.05 and ** P < 0.01

Article Snippet: The wild-type expression plasmid pCMV-AURKB (NM_004217.2) was obtained from SinoBiological (Beijing, China).

Techniques: Expressing, CCK-8 Assay, Western Blot

Journal: Human Cell

Article Title: Role of aurora kinase B in regulating resistance to paclitaxel in breast cancer cells

doi: 10.1007/s13577-022-00675-8

Figure Lengend Snippet: Effect of the phosphorylation level of AURKB on PTX resistance of breast cancer cells. a , b The relative expression levels of AURKB mRNA and protein in MDA-MB-231/PTX-resistant and AURKB stable knockdown cells (231/PTX-A) by quantitative PCR and Western blot. c , d The relative expression levels of AURKB mRNA and protein in AURKB stable knockdown cells (231/PTX-A) transfected with wild-type and mutant-type expression plasmids for 48 h by quantitative PCR and Western blot. e Detection of the survival rate in different groups at different time points using CCK-8 method after cell treatment with PTX (0.5 μM); f , g The cells labeled with luciferase were xenografted subcutaneously into NOD-SCID mice. At the fourth week, the fluorescence intensity of subcutaneous tumor was examined by in vivo imaging. One-way ANOVA with Tukey post hoc analysis. * P < 0.05 and ** P < 0.01

Article Snippet: The wild-type expression plasmid pCMV-AURKB (NM_004217.2) was obtained from SinoBiological (Beijing, China).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Mutagenesis, CCK-8 Assay, Labeling, Luciferase, Fluorescence, In Vivo Imaging

Journal: Human Cell

Article Title: Role of aurora kinase B in regulating resistance to paclitaxel in breast cancer cells

doi: 10.1007/s13577-022-00675-8

Figure Lengend Snippet: Effect of phosphorylation on the cellular localization of AURKB. a Analysis of the distribution differences of AURKB in the cytoplasm (normalized to GAPDH) and nucleus (normalized to Lamin B1) of 231 and 231/PTX cells using Western blot after the extraction of cytoplasm and nucleus, respectively. b Analysis of the distribution differences of AURKB in the cytoplasm and nucleus of BT549 and BT549/PTX cells using Western blot. c Analysis of the distribution differences of AURKB in the cytoplasm and nucleus after 48 h of DMSO and bisindolylmaleimide I (1 μM) treatment of 231/PTX cells using Western blot analysis. d Analysis of the subcellular localization of wild-type and mutant-type AURKB using immunofluorescence after the transfection of overexpression plasmid of AURKB in 231/PTX-A cells with stable knockdown of AURKB for 48 h. Scale bar, 20 μm. e The distribution difference of wild-type and mutant-type AURKB in cytoplasm and nucleus using Western blot after the transfection of overexpression plasmid of AURKB in 231/PTX-A cells for 48 h

Article Snippet: The wild-type expression plasmid pCMV-AURKB (NM_004217.2) was obtained from SinoBiological (Beijing, China).

Techniques: Western Blot, Mutagenesis, Immunofluorescence, Transfection, Over Expression, Plasmid Preparation

Journal: Human Cell

Article Title: Role of aurora kinase B in regulating resistance to paclitaxel in breast cancer cells

doi: 10.1007/s13577-022-00675-8

Figure Lengend Snippet: Correlation analysis of AURKB and RAB27B expression. a Correlation analysis between AURKB mRNA and RAB27B mRNA expression using online tool UALCAN to collect breast cancer data in TCGA. b , c Analysis of RAB27B mRNA and protein expression differences in breast cancer cells MDA-MB-231 and corresponding PTX-resistant MDA-MB-231/PTX cells using quantitative PCR and Western blot. d , e Analysis of the mRNA and protein expression of RAB27B using PCR and Western blot after the transfection with wild-type or mutant-type AURKB expression plasmids in 231/PTX-A cells with stable AURKB knockdown, and simultaneous treatment with DMSO or Bisindolylmaleimide I (1 μM) for 24 h. One-way ANOVA with Tukey post hoc analysis. * P < 0.05 and ** P < 0.01

Article Snippet: The wild-type expression plasmid pCMV-AURKB (NM_004217.2) was obtained from SinoBiological (Beijing, China).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Mutagenesis

Journal: Human Cell

Article Title: Role of aurora kinase B in regulating resistance to paclitaxel in breast cancer cells

doi: 10.1007/s13577-022-00675-8

Figure Lengend Snippet: Interaction between AURKB and RAB27B. a Detection of the interaction between AURKB and RAB27B by immunoprecipitation assay using DDK-labeled antibody after the transfection with wild-type or mutant-type AURKB expression plasmids in 231/PTX-A cells with stable AURKB knockdown. b Detection of the relative content of RAB27B protein at different time points after the transfection of 231/PTX-A with wild-type or mutant-type AURKB expression plasmid for 24 h, and subsequent treatment with Cycloheximide (100 μg/mL). c Quantification of RAB27B protein levels (normalized to β-actin) at different time points. One-way ANOVA with Tukey post hoc analysis. * P < 0.05 and ** P < 0.01

Article Snippet: The wild-type expression plasmid pCMV-AURKB (NM_004217.2) was obtained from SinoBiological (Beijing, China).

Techniques: Immunoprecipitation, Labeling, Transfection, Mutagenesis, Expressing, Plasmid Preparation