Journal: International Journal of Clinical and Experimental Pathology

Article Title: Effects of mitochondrial uncoupling protein 2 inhibition by Genipin on rat bone marrow mesenchymal stem cells under hypoxia and serum deprivation (H/SD) conditions

doi:

Figure Lengend Snippet: Effects of UCP2 inhibited by Genipin on apoptosis related protein. (A) The Bax, Bcl2, caspase-3 and cleaved caspase-3 protein were quantified by Western blotting. The protein levels of Bax (B), Bcl2 (C), caspase-3 (D) and cleaved caspase-3 (E) were normalized to β-actin. Data are expressed as the mean ± SE of three independent experiments. Different letters indicated significantly different at P<0.05.

Article Snippet: The primary antibody to β-actin was used at a concentration of 1:1000 (Bioss, China).

Techniques: Western Blot

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 ) Primary cells were extracted from breast cancer tissues and adjacent normal breast tissues. The cytoplasmic and nuclear protein lysates representing an equal number of cells were subjected to immunoblot (IB) analysis. ( b ) Representative IHC staining showing AURKA expression. Images were magnified with a × 4 or × 40 objective. Scale bar, 50 μm. ( c ) Mouse embryonic fibroblasts (MEFs) overexpressing K-Ras, H-Ras and the vector control (Vec) were analysed by IB for indicated antibodies (left panel). Cytoplasmic and nuclear lysates of WT (−)-, vector control (Vec)-, K-Ras- and H-Ras-overexpressed MEFs were subjected to IB analysis (right panel). ( d ) MDA-MB-231, SUM149 or BT549 cells were treated with AURKA siRNAs for 96 h. Adherent cells were collected for IB analysis (left panel), CD24/CD44 staining and CD24 low /CD44 high population analysis via flow cytometry (right panel). ( e ) HA-tagged AURKA was overexpressed in MCF-10A, SUM149 and BT549 cells through lentivirus-mediated gene transfer. Puromycin (1 μg ml −1 ) selected cells were collected for IB analysis (left panel), CD24/CD44 staining and CD24 low /CD44 high population analysis by using flow cytometry (right panel). ( f ) Gene expression data acquired from a public database (GEO ID: GSE23541, the group of AURKA+Dox and AURKA-Dox) were subjected to GSEA using the gene expression signature that was downregulated in the CD24 low /CD44 high -sphere population. ( g ) MDA-MB-231 cells were co-transfected with shRNA vector (sh Control or shAURKA) and the overexpression vector (ER or AER) via lentivirus-mediated gene transfer. Puromycin (1 μg ml −1 ) and blasticidin (2 μg ml −1 ) double selected cells were subjected to CD24/CD44 staining and analysis via flow cytometry. ( h ) Cells derived from g were subjected to mammosphere culture assay (6 days, upper panel) and the secondary passaging (additional 6 days, lower panel). Scale bar, 100 μm. ( i ) Data from three independent experiments derived from g were used for quantitative analysis of mammosphere size (diameter, Φ) and mammosphere number. Left panel shows distribution pattern of mammosphere size from MDA-MB-231 (Kruskal–Wallis test followed by Dunn's multiple comparison test, *** P <0.001). Right panel shows the number of mammospheres (Φ>60 μm). 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: The following primary antibodies were used: AURKA (rabbit; Millipore, 1:4,000, catalogue number 07–648), AURKA (mouse; Abcam, 1:4,000, catalogue number ab13824), phospho-AURKA (T288; Cell Signaling, 1:1,000, catalogue number 3079), haemagglutinin (HA) tag (Sigma-Aldrich, 1:4,000, catalogue number SAB4300603), FLAG tag (Sigma-Aldrich, 1:4,000, catalogue number F1804), glyceraldehyde 3-phosphate dehydrogense (Thermo Fisher Scientific, 1:4,000, catalogue number AM4300), β-actin (Cell Signaling, 1:4,000, catalogue number 4967), c-Myc (Santa Cruz, 1:2,000, catalogue number sc-764), Histone H3 (Cell Signaling, 1:2,000, catalogue number 4499), hnRNP K (Cell Signaling, 1:4,000, catalogue number 4675), enhanced green fluorescent protein (Cell Signaling, 1:4,000, catalogue number 2555), GST (Cell Signaling, 1:4,000, catalogue number 2624s), Erk1/2 (Cell Signaling, 1:2,000, catalogue number 9102), phospho-Erk1/2 (Thr202/Tyr204; Cell Signaling, 1:2,000, catalogue number 9101), Ras (Cell Signaling, 1:1,000, catalogue number 3965), phospho-p53 (Ser315; Cell Signaling, 1:1,000, catalogue number 2528), p53 (Cell Signaling, 1:1,000, catalogue number 2524s), phospho-Histone H3 (Ser10; Cell Signaling, 1:1,000, catalogue number 9701), Histone H3 (Cell Signaling, 1:1,000, catalogue number 9715).

Techniques: Western Blot, Immunohistochemistry, Expressing, Plasmid Preparation, Control, Staining, Flow Cytometry, Gene Expression, Transfection, shRNA, Over Expression, Derivative Assay, Passaging, Comparison

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: The following primary antibodies were used: AURKA (rabbit; Millipore, 1:4,000, catalogue number 07–648), AURKA (mouse; Abcam, 1:4,000, catalogue number ab13824), phospho-AURKA (T288; Cell Signaling, 1:1,000, catalogue number 3079), haemagglutinin (HA) tag (Sigma-Aldrich, 1:4,000, catalogue number SAB4300603), FLAG tag (Sigma-Aldrich, 1:4,000, catalogue number F1804), glyceraldehyde 3-phosphate dehydrogense (Thermo Fisher Scientific, 1:4,000, catalogue number AM4300), β-actin (Cell Signaling, 1:4,000, catalogue number 4967), c-Myc (Santa Cruz, 1:2,000, catalogue number sc-764), Histone H3 (Cell Signaling, 1:2,000, catalogue number 4499), hnRNP K (Cell Signaling, 1:4,000, catalogue number 4675), enhanced green fluorescent protein (Cell Signaling, 1:4,000, catalogue number 2555), GST (Cell Signaling, 1:4,000, catalogue number 2624s), Erk1/2 (Cell Signaling, 1:2,000, catalogue number 9102), phospho-Erk1/2 (Thr202/Tyr204; Cell Signaling, 1:2,000, catalogue number 9101), Ras (Cell Signaling, 1:1,000, catalogue number 3965), phospho-p53 (Ser315; Cell Signaling, 1:1,000, catalogue number 2528), p53 (Cell Signaling, 1:1,000, catalogue number 2524s), phospho-Histone H3 (Ser10; Cell Signaling, 1:1,000, catalogue number 9701), Histone H3 (Cell Signaling, 1:1,000, catalogue number 9715).

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 , b and c ) Primary breast cancer cells were isolated from breast cancer tissues derived from ten patients. Nuclear and cytoplasmic fractions were subjected to immunoblotting (IB) analysis ( a ). The expression of CD24 and CD44 were analysed using flow cytometry ( b ). Nuclear and cytoplasmic fractions of AURKA were normalized using histone H3 and β-actin, respectively. The nuclear fraction of c-Myc and hnRNP K were normalized using histone H3. The normalized AURKA, c-Myc, hnRNP K and the CD24 low /CD44 high population derived from the same patient were used to compose a scatterplot and linear regression analysis performed ( c ). ( d ) Breast cancer tissues were subjected to IHC staining for indicated antibodies. Representative images were acquired with × 10 and × 40 objectives (upper panel). Scale bar, 50 μm. Pearson's χ 2 -test was used to analyse the correlation between nuclear AURKA and c-Myc/CD24 (lower panel). ( e ) IHC staining for AURKA and c-Myc was performed on breast cancer tissues. Kaplan–Meier analysis was performed and log-rank test used to make statistical comparisons. ( f ) During breast cancer development, AURKA is overexpressed and translocates to the nucleus, where the nuclear AURKA acts as a transactivating factor that binds and activates the MYC promoter through its interaction with hnRNP K. Through these mechanisms, nuclear AURKA enhances BCSC phenotype. Importantly, these processes are dependent on the nuclear localization of AURKA rather than its kinase activity. Traditional targeted therapies focus on the inhibition of kinase activity, which may be insufficient for suppressing the kinase-independent oncogenic actions. These new findings suggest that targeting the spatial deregulation of kinase could be a promising strategy for overcoming kinase inhibitor insensitivity.

Article Snippet: The following primary antibodies were used: AURKA (rabbit; Millipore, 1:4,000, catalogue number 07–648), AURKA (mouse; Abcam, 1:4,000, catalogue number ab13824), phospho-AURKA (T288; Cell Signaling, 1:1,000, catalogue number 3079), haemagglutinin (HA) tag (Sigma-Aldrich, 1:4,000, catalogue number SAB4300603), FLAG tag (Sigma-Aldrich, 1:4,000, catalogue number F1804), glyceraldehyde 3-phosphate dehydrogense (Thermo Fisher Scientific, 1:4,000, catalogue number AM4300), β-actin (Cell Signaling, 1:4,000, catalogue number 4967), c-Myc (Santa Cruz, 1:2,000, catalogue number sc-764), Histone H3 (Cell Signaling, 1:2,000, catalogue number 4499), hnRNP K (Cell Signaling, 1:4,000, catalogue number 4675), enhanced green fluorescent protein (Cell Signaling, 1:4,000, catalogue number 2555), GST (Cell Signaling, 1:4,000, catalogue number 2624s), Erk1/2 (Cell Signaling, 1:2,000, catalogue number 9102), phospho-Erk1/2 (Thr202/Tyr204; Cell Signaling, 1:2,000, catalogue number 9101), Ras (Cell Signaling, 1:1,000, catalogue number 3965), phospho-p53 (Ser315; Cell Signaling, 1:1,000, catalogue number 2528), p53 (Cell Signaling, 1:1,000, catalogue number 2524s), phospho-Histone H3 (Ser10; Cell Signaling, 1:1,000, catalogue number 9701), Histone H3 (Cell Signaling, 1:1,000, catalogue number 9715).

Techniques: Isolation, Derivative Assay, Western Blot, Expressing, Flow Cytometry, Immunohistochemistry, Activity Assay, Inhibition

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Role of Estrogen Receptor-Positive/Negative Ratios in Regulating Breast Cancer

doi: 10.1155/2022/7833389

Figure Lengend Snippet: The ER α +/ER α − ratio plays a critical role in the tumor microenvironment. (a) 1/2: M2 type macrophage polarization at different ER expression levels in tumor microenvironment. M2 macrophage polarization was induced significantly ( P < 0.05) in the 70% ER α +/ER α − ratio group. (b) BRCA1 and HER-2 expressing in tumor tissue. BRCA1 and HER-2 expression were induced significantly ( P < 0.05) in 40% and 70% ER α +/ER α − ratio groups.

Article Snippet: The membranes were blocked for 1 h at 26°C with 5% bovine serum albumin containing 0.1% Tween-20, incubated with the primary antibodies (breast cancer gene 1 (BRCA1) and HER2, Beyotime, China; 1 : 1000) overnight at 4°C.

Techniques: Expressing

Journal: Biomedicines

Article Title: Melatonin Modulation of Radiation-Induced Molecular Changes in MCF-7 Human Breast Cancer Cells

doi: 10.3390/biomedicines10051088

Figure Lengend Snippet: Effects of melatonin and radiation on breast cancer miRNA expression microarray. Total RNA from MCF-7 cells was extracted 4 h after radiation, reverse transcribed and used for RT-PCR analysis using Human Breast Cancer microarray (MIHS-109ZA). ( a – c ) Heatmaps of relative normalized expression between different treatments ( a ) M vs. C; ( b ) R vs. C; ( c ) M + R vs. C; ( d ) Bar chart of relative normalized expression (ΔCt) of selected miRNAs. a: miR20a; b: miR-20b; c: miR-17; d: miR-141; e: miR-15a; f: miR-19a; g: miR29a; h: miR-93; i: miR-10b. C: Control; M: Melatonin pre-treated cells (1 nM); R: Radiated cells (8 Gy); M + R: Melatonin pre-treated and radiated cells.

Article Snippet: We next analyzed the influence of melatonin on the expression of multiple genes and miRNAs involved in breast cancer using a gene microarray (Human Breast Cancer RT 2 Profiler TM PCR Array) and a Human Breast Cancer miRNA microarray (MIHS-109ZA, Qiagen, Germantown, MD, USA).

Techniques: Expressing, Microarray, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Biomedicines

Article Title: Melatonin Modulation of Radiation-Induced Molecular Changes in MCF-7 Human Breast Cancer Cells

doi: 10.3390/biomedicines10051088

Figure Lengend Snippet: Changes in miRNA expression levels in MCF-7 cells. Cells were pre-treated with 1 nM melatonin for 1 week prior to being irradiated (8 Gy), and after 4 h total RNA was isolated and reverse transcribed; ( a ) RT-PCR analysis of selected miRNAs (miR-20a, miR-19a, miR-93, miR-20b, miR-29a, miR10a and miR10b) where the pre-treatment with melatonin potentiated the inhibitory effect of radiation; ( b ) RT-PCR analysis of selected miRNAs (miR-17, miR-141 and miR-15a) induced by radiation and counteracted by melatonin pre-treatment; ( c ) RT-PCR analysis of miR34a stimulated by radiation and melatonin. Data are expressed as relative normalized expression compared to control cells (mean ± S.E.M.). a, p ≤ 0.001 vs. C; b, p ≤ 0.001 vs. R; c, p ≤ 0.05 vs. R; d, p ≤ 0.01 vs. R.

Article Snippet: We next analyzed the influence of melatonin on the expression of multiple genes and miRNAs involved in breast cancer using a gene microarray (Human Breast Cancer RT 2 Profiler TM PCR Array) and a Human Breast Cancer miRNA microarray (MIHS-109ZA, Qiagen, Germantown, MD, USA).

Techniques: Expressing, Irradiation, Isolation, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Control

Journal: eLife

Article Title: Differential translation of mRNA isoforms underlies oncogenic activation of cell cycle kinase Aurora A

doi: 10.7554/eLife.87253

Figure Lengend Snippet: ( A ) Complementarity of hsa-let-7a binding to Aurora Kinase A (AURKA) 3′UTR. ( B ) Mean and SEM of median Venus/mCherry mean fluorescence intensity (MFI) ratios from U2OS cells co-transfected with 250 nM hsa-let-7a or a negative control (NC) miRNA from three biological replicates. n ≥ 182 cells per condition. Unpaired t -test. ( C ) Same as ( B ) but co-transfecting 300 nM anti-let-7a or NC. n ≥ 94 cells per condition. Unpaired t -test. ( D ) RT-qPCR of reporter mRNAs abundance from U2OS cells transfected as ( B ), at 8 hr of 10 μg/ml ActD. mCherry mRNA used as reference target. Ordinary one-way ANOVA with Tukey’s multiple-comparisons test. ( E ) Translation rate imaging by rate of protein stabilization (TRIPS) (left) and C-TRIPS (right) assays in transfected U2OS CDK2 cells. n ≥ 162 cells per condition Left: mean and SEM from three biological replicates. Ordinary one-way ANOVA with Dunnett’s multiple-comparisons test vs. NC. Right : median and 95% CI of pooled data from left. Kruskal–Wallis with Dunnett’s multiple-comparisons test vs. NC of the respective phase. ns, not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Figure 5—source data 1. Numerical data for graphs.

Article Snippet: Primary antibodies were mouse anti-AURKA (1:1000; Clone 4/IAK1, BD Transduction Laboratories), rabbit anti-GFP (1:5000; ab290, Abcam), rabbit anti-GAPDH (1:4000; 2118S, CST), rabbit anti-mCherry (1:1000; ab167453, Abcam), mouse anti-Flag M2 (1:1000; F1804, Sigma).

Techniques: Binding Assay, Fluorescence, Transfection, Negative Control, Quantitative RT-PCR, Imaging