Journal: BMC Cancer

Article Title: A genomics approach to identify susceptibilities of breast cancer cells to “fever-range” hyperthermia

doi: 10.1186/1471-2407-14-81

Figure Lengend Snippet: List of the top and bottom most regulated genes for each cell line in the C vs H and C’ vs H’ comparisons

Article Snippet: MCF10A (ATCC #CRL10318) mammary epithelial cells and MCF7 luminal breast cancer cells (ATCC #HTB-22), MDA-MB-231 Basal B breast cancer cells (ATCC # HTB-26), and MDA-MB-468 Basal A breast cancer cells (ATCC #HTB-132) were purchased from ATCC and grown in standard culture conditions as previously reported [ - ].

Techniques: Binding Assay, Sterility, Sequencing

Journal: BMC Cancer

Article Title: A genomics approach to identify susceptibilities of breast cancer cells to “fever-range” hyperthermia

doi: 10.1186/1471-2407-14-81

Figure Lengend Snippet: qPCR and microarray expression data for selected cell cycle genes in the H’ vs H comparison

Article Snippet: MCF10A (ATCC #CRL10318) mammary epithelial cells and MCF7 luminal breast cancer cells (ATCC #HTB-22), MDA-MB-231 Basal B breast cancer cells (ATCC # HTB-26), and MDA-MB-468 Basal A breast cancer cells (ATCC #HTB-132) were purchased from ATCC and grown in standard culture conditions as previously reported [ - ].

Techniques: Microarray, Expressing

Journal: The Journal of Clinical Investigation

Article Title: Six1 expands the mouse mammary epithelial stem/progenitor cell pool and induces mammary tumors that undergo epithelial-mesenchymal transition

doi: 10.1172/JCI37691

Figure Lengend Snippet: (A) Breast tumor tissue microarrays were subjected to immunohistochemistry using antibodies against Six1 and cyclin D1. They were then scored for staining intensity on a 0–4 scale. Representative examples are shown of tumor cores with low-intensity Six1 staining ([scored 0–1], core number AA6), medium-intensity Six1 staining ([scored 1.5–2.5], core number DB6), and high-intensity Six1 staining ([scored 3–4], core number DB8), with their corresponding intensity of cyclin D1 staining (original magnification, ×40). (B) The intensity of cyclin D1 staining increases with increasing intensity of Six1 staining, as scored on a 0–4 scale. Six1 staining intensity groups were established as described in A. Error bars represent mean ± SEM.

Article Snippet: High-density breast cancer tissue microarrays were obtained from Biomax (BR1921), and immunohistochemistry was performed using the protocols detailed above for Six1 (Atlas Antibodies) and cyclin D1 (Thermo Scientific).

Techniques: Immunohistochemistry, Staining

Journal: American Journal of Cancer Research

Article Title: Upregulation of CYP1B1 by hypoxia is mediated by ERα activation in breast cancer cells

doi:

Figure Lengend Snippet: Analysis of HIF-1α and CYP1B1 expression in tissue microarray (TMA) samples from breast cancer patients. (A) Schematic diagram of the tumor stages of each breast tissue core. (B) Hematoxylin and eosin (H&E) staining images provided by the manufacturer. Immunofluorescence images of tumor microarrays (TMA) stained with anti-hypoxia-inducible factor-1 alpha (HIF-1α) (C) and anti-cytochrome P450 1B1 (CYP1B1) (D) antibodies. Images were acquired at 20X magnification using a Leica Thunder microscope. Whole tissue slide images were mosaic merged via LAS-X software. (E) Representative images of immunofluorescence analysis of HIF-1α (green) and CYP1B1 (green) in two slides of ER-negative and positive breast cancer TMA. Immunofluorescence images were acquired using a Leica Thunder microscope. (F) Scatter plots showing fluorescence intensity correlation analysis of HIF-1α and CYP1B1 in 64 ER-positive TMAs. (G) Violin plots of fluorescence intensities of HIF-1α and CYP1B1. Samples were classified into low (immunohistochemical [IHC] score: 1+, 2+) and high (IHC score: 3+) groups according to the estrogen receptor (ER) expression status of the TMA specification sheet, and the correlation between HIF-1α and CYP1B1 expression was analyzed. (H) Analysis of fluorescence intensity of CYP1B1 against HIF1α expression in ER-positive TMA. The fluorescence intensity of CYP1B1 was analyzed by classifying the fluorescence intensity of HIF1-α based on the top and bottom 25% ER expression. *, P<0.0001 versus ER-positive tumor tissue with low HIF1-α fluorescence intensity by Mann-Whitney test.

Article Snippet: Immunofluorescence analysis of tumor microarrays (TMAs) A human breast cancer tissue microarray (TMA, #BC081120e) containing 110 cases was purchased from Biomax (Rockville, MD).

Techniques: Expressing, Microarray, Staining, Immunofluorescence, Microscopy, Software, Fluorescence, Immunohistochemical staining, MANN-WHITNEY

Journal: Oncogene

Article Title: Nuclear Aurora-A kinase-induced hypoxia signaling drives early dissemination and metastasis in breast cancer: implications for detection of metastatic tumors

doi: 10.1038/s41388-021-01969-1

Figure Lengend Snippet: A. Representative images of tissue microarray IHC, (n = 206) stained with AURKA/DAB-brown, hematoxylin-nuclei/blue. Scale bar-300 μm. Insets-x250 enlarged areas. B, C Quantification N-AURKA positive(+) cells as in A, 3 randomly-assigned fields, n = 100 cells/field. B Pathological stages (Normal = 32, DCIS = 24, IDC = 72, MIDC-LN = 32, ILC = 24) and C receptor-based subtypes (Normal = 32, TNBC = 39, HER2+ = 32, ER+/PR+ = 30). D WB analysis of nuclear/cytoplasmic fractionations, as indicated. E Quantification of AURKA in cytoplasm/nucleus, percent-of-total, normalized to controls. F Schematic outline of cell line production. G, H Immunofluorescence and WB analysis of AURKA-sublines produced in F stained with AURKA (green), RFP(red), DAPI-nuclei/blue; clones indicated as c1/c2/c3. Scale bar-10 μm. One-way ANOVA, ±S.E.M, Dunnett’s test, ns non-significant.

Article Snippet: Chromatin immunoprecipitation (ChIP) was performed using ChIP-grade antibodies against human AURKA (BethylLabs, #IHC-00062) and the SimpleChIP Enzymatic ChromatinIP Kit (CellSignaling) according to manufacturer’s instructions.

Techniques: Microarray, Staining, Immunofluorescence, Produced, Clone Assay

Journal: Oncogene

Article Title: Nuclear Aurora-A kinase-induced hypoxia signaling drives early dissemination and metastasis in breast cancer: implications for detection of metastatic tumors

doi: 10.1038/s41388-021-01969-1

Figure Lengend Snippet: A Experimental design of xenograft study, n = 3 mice/group. B Quantification of Fluorescent-IHC using phospho-AURKA-T288 antibody in tumors. n = 50 mitotic-cells/tumor. C Quantification of final tumor volume/mm3. D Representative H&E images of lung metastases and DAB-IHC images of liver metastases stained with anti-RFP-antibody. Metastases outlined by black line. Scale bar-50 μm. E, F Quantification of metastases: number and area of metastases normalized to total area. G Lung and liver metastasis penetrance in mice treated with vehicle or MLN8237. One-way ANOVA, ±S.E.M. Tukey’s test. H Model of Nuclear-AURKA-HIF1 mediated gene expression. Normoxia: HIF1A is hydroxylated/ubiquitinated by PHDs/VHL resulting in degradation. Hypoxia: low-oxygen stabilizes HIF1A leading to HIF1A/B dimerization/activation, transactivation of hypoxia-response genes. Normoxia+N-AURKA: HIF1A/B activity is increased leading to metastasis. MLN8237 inhibits AURKA activity decreasing metastasis.

Article Snippet: Chromatin immunoprecipitation (ChIP) was performed using ChIP-grade antibodies against human AURKA (BethylLabs, #IHC-00062) and the SimpleChIP Enzymatic ChromatinIP Kit (CellSignaling) according to manufacturer’s instructions.

Techniques: Staining, Gene Expression, Activation Assay, Activity Assay

Journal: Oncogene

Article Title: Nuclear Aurora-A kinase-induced hypoxia signaling drives early dissemination and metastasis in breast cancer: implications for detection of metastatic tumors

doi: 10.1038/s41388-021-01969-1

Figure Lengend Snippet: A WB analysis of HIFs in cells with indicated antibodies, DMSO-vehicle or DMOG for 7 h. B Quantification of WB results as in A, fold of change over DMSO-control. C, D Representative images of Immunoprecipitation/WB analysis with indicated antibodies, WCL-whole cell lysate. E Quantification of ChIP qPCR against selected promoter region, normalized to total input. F WB analysis of ChIP (before de-crosslinking). G Mass-spectrometry analysis of AURKA-IP complexes: Venn diagram displaying the numbers of proteins found in the NLS- or NES-AURKA complexes. The 850 proteins/yellow were further filtered, nuclear/blue. Pie-chart showing distribution of N-AURKA binding partners based on functional Panther Gene Ontology terms. Tables showing selected nuclear protein classifications for H RNA-binding and I DNA-binding from PANTHER Gene Ontology. One-way ANOVA, ±S.E.M, Dunnett’s test, ns non-significant.

Article Snippet: Chromatin immunoprecipitation (ChIP) was performed using ChIP-grade antibodies against human AURKA (BethylLabs, #IHC-00062) and the SimpleChIP Enzymatic ChromatinIP Kit (CellSignaling) according to manufacturer’s instructions.

Techniques: Control, Immunoprecipitation, ChIP-qPCR, Mass Spectrometry, Binding Assay, Functional Assay, RNA Binding Assay

Journal: Oncogene

Article Title: Nuclear Aurora-A kinase-induced hypoxia signaling drives early dissemination and metastasis in breast cancer: implications for detection of metastatic tumors

doi: 10.1038/s41388-021-01969-1

Figure Lengend Snippet: A Heat-map of differentially expressed genes (DEGs, mean values) in cells. Gene expression (n = 3167) is normalized log2 counts/million. B Principal Component Analysis of RNA-Seq libraries as in A. C Volcano-plot analysis of RNAseq data as in A NLS-AURKA vs. control, log2 fold changes of gene expression on the x-axis, FDR statistical significance (−log10 p value) on the y-axis; upregulated (red), downregulated (black) genes in NLS, all genes (blue). Genes with at least a >1.5 fold change and FDR < 0.01 are displayed. D Visualization of Gene Ontology terms for NLS-AURKA vs. control; up/downregulated GOterms (FDR < 0.1) are depicted as circles; the distance indicates the relationship between terms: closer distance means higher similarity. Color and size of circles indicate significance of differential expression of an individual GO term in log10 p value. E WB analysis of selected RNA-seq target proteins as in A. F Quantification of WB in E, fold change over control. G GSEA comparison NLS-AURKA vs. control for selected GOterm gene sets. H Heat-map of HIF-dependent DEGs. I Predicted upstream regulators: activated (yellow), inhibited (blue) using IPA activation z-score. One-way ANOVA, ±S.E.M, Dunnett’s test, ns non-significant.

Article Snippet: Chromatin immunoprecipitation (ChIP) was performed using ChIP-grade antibodies against human AURKA (BethylLabs, #IHC-00062) and the SimpleChIP Enzymatic ChromatinIP Kit (CellSignaling) according to manufacturer’s instructions.

Techniques: Gene Expression, RNA Sequencing, Control, Quantitative Proteomics, Comparison, Activation Assay

Journal: Oncogene

Article Title: Nuclear Aurora-A kinase-induced hypoxia signaling drives early dissemination and metastasis in breast cancer: implications for detection of metastatic tumors

doi: 10.1038/s41388-021-01969-1

Figure Lengend Snippet: A WB analysis of HIFs in cells as indicated, treated with siRNA: control-scr or anti-HIF1A/B. B Quantification of WB results in A, fold of change over siScr. C Individual cell movement tracking-plots toward chemoattractant. Cell lines as indicated. Graphs of D total distance, E cell-body directionality, F cell speed. G qPCR analysis of select genes in control and NLS-AURKA cells with siScr and siHIF1A/B, fold change over control-siScr. H qPCR analysis of FOXM1 in control and NLS-AURKA cells treated with siScr and siHIF1A/B, fold change over control-siScr. I, J qPCR analysis of FOXM1, HIF1A, and HIF1B in control and NLS-AURKA cells treated with siScr and siFOXM1, fold change over control-siScr. HIF1B: (unpaired t test: con-Scr vs NLS-AURKA-Scr). One-way ANOVA, ±S.E.M. Tukey’s test, ns non-significant.

Article Snippet: Chromatin immunoprecipitation (ChIP) was performed using ChIP-grade antibodies against human AURKA (BethylLabs, #IHC-00062) and the SimpleChIP Enzymatic ChromatinIP Kit (CellSignaling) according to manufacturer’s instructions.

Techniques: Control