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

Journal: bioRxiv

Article Title: Oncogenic PKA signaling stabilizes MYC oncoproteins via an aurora kinase A-dependent mechanism

doi: 10.1101/2021.04.16.438110

Figure Lengend Snippet: (A) Colo741 and FLX1 cells in 96-well plates were treated with various types of AURKA inhibitors for 72h at different concentrations, and their relative cell viability was measured by CTG assay versus untreated control samples. Results are the mean ±s.e.m of triple biological replicates, three technical replicates per biological replicate. Inhibitors are colored based on their binding mode. (B) Immunoblots showing the expression of c-MYC and n-MYC in Colo741 and FLX1 cells after treating with CD532 or MLN8237 for 24 hours. (C) RT-qPCR analysis of MYC, MYCN and their downstream genes in FLX1 cells treated with 1uM CD532 for 24 hours. Results are the mean ±s.e.m of three biological replicates, three technical replicates for each biological replicate, targets with P value <0.05 form paired t-test for CD532 treated versus DMSO were labelled with the asterisk. (D) Immunoblots showing the change of c-MYC and n-MYC levels in Colo741 and FLX1 cells after MYC or MYCN siRNA knockdown for 48 hours. (E) Immunoblots showing the induction of Tet-on 3xFLAG- MYC and MYCN in respective FLX1 transgenic cells after dox induction for 48 hours. (F) Relative confluence of Colo741 and FLX1 cells in 96 well plates after MYC or MYCN knockdown with siRNA. FLX1 cells were incubated 36 hours before recording to ensure better adhesion. Experiments were done in duplicate and representative results were shown with mean ±s.d, n = 10 for each condition. (G) Relative confluence of transgenic FLX1 cells with doxycycline-controlled Tet-on 3xFLAG- MYC or MYCN in 96 well plates after treatment with or without 1ug/ml dox. Confluence was imaged for 120 hours and analyzed by Incucyte. Experiment was duplicate, the representative results shown with mean ±s.d, n = 6 for each condition.

Article Snippet: Blocked membranes were immunoblotted with antibodies against the following targets separately: AURKA (CST #14475, Biolegend #603301), Phospho-PKA Substrate (CST#9624), c-MYC (CST#18583), n-MYC (CST#84406), PIM1 (CST#3247), PIM2 (CST#4730), PKAC-α (CST#4782), PKAR1a (CST#5675), FLAG (Sigma#F1804), Actin (CST#3700) or COXIV (CST#5247).

Techniques: CTG Assay, Binding Assay, Western Blot, Expressing, Quantitative RT-PCR, Transgenic Assay, Incubation

Journal: bioRxiv

Article Title: Oncogenic PKA signaling stabilizes MYC oncoproteins via an aurora kinase A-dependent mechanism

doi: 10.1101/2021.04.16.438110

Figure Lengend Snippet: (A) siRNA kinase library screen with FLX1 in 384 well plates for 7 days shows the effect of each target kinase on cell proliferation (average of 3 biological replicates). Selected non-metabolic kinases that decrease cell proliferation with z-score < -1 were marked. (B) Immunoblots showing the change of c-MYC and n-MYC levels in Colo741 and FLX1 cells after PIM1 or PIM2 siRNA knockdown for 48 hours. (C) Immunoblot showing the change of PKA activity, as indicated by phospho-PKA substrate, and c-MYC and n-MYC levels in Colo741 and FLX1 cells after treating with DMSO, 1uM CD532, 1uM MLN8237, 1uM CX6258 or combination of 1uM MLN8237 and 1uM CX6258 for 24 hours. (D) Immunoblots showing the change of PIM2 expression in Colo741 and FLX1 cells after PRKACA siRNA knockdown for 24, 48 and 72 hours and NTC for 72 hours. (E) Immunoblots showing the change of PIM2 level after MYC or MYCN overexpression in Colo741 or FLX1 transgenic cells after dox induction for 48 hours. (F) Immunoblots showing the change of PIM2 levels in Colo741 and FLX1 cells after MYC or MYCN siRNA knockdown for 48 hours. (G) Schematic of DNAJ-PKAc mediating cell proliferation in FLC by stabilizing MYC family proteins via AURKA and producing a positive feedback loop with PIM2.

Article Snippet: Blocked membranes were immunoblotted with antibodies against the following targets separately: AURKA (CST #14475, Biolegend #603301), Phospho-PKA Substrate (CST#9624), c-MYC (CST#18583), n-MYC (CST#84406), PIM1 (CST#3247), PIM2 (CST#4730), PKAC-α (CST#4782), PKAR1a (CST#5675), FLAG (Sigma#F1804), Actin (CST#3700) or COXIV (CST#5247).

Techniques: Western Blot, Activity Assay, Expressing, Over Expression, Transgenic Assay

Journal: bioRxiv

Article Title: Oncogenic PKA signaling stabilizes MYC oncoproteins via an aurora kinase A-dependent mechanism

doi: 10.1101/2021.04.16.438110

Figure Lengend Snippet: (A) Immunoblots showing the basal level of PKA activity in phosphorylated PKA substrate and c-MYC, n-MYC, and PIM2 in AML12 WT (left) and AML12 DNAJ-PKAc cells (right). (B) Immunoblot showing the presence of DNAJ-PKAc and different level of c-MYC, n-MYC, AURKA and PIM2 in FLC tumor samples (FLC) versus adjacent liver (N) from 4 FLC patients.

Article Snippet: Blocked membranes were immunoblotted with antibodies against the following targets separately: AURKA (CST #14475, Biolegend #603301), Phospho-PKA Substrate (CST#9624), c-MYC (CST#18583), n-MYC (CST#84406), PIM1 (CST#3247), PIM2 (CST#4730), PKAC-α (CST#4782), PKAR1a (CST#5675), FLAG (Sigma#F1804), Actin (CST#3700) or COXIV (CST#5247).

Techniques: Western Blot, Activity Assay

Journal: Human Molecular Genetics

Article Title: Functional characterization of a chr13q22.1 pancreatic cancer risk locus reveals long-range interaction and allele-specific effects on DIS3 expression

doi: 10.1093/hmg/ddw300

Figure Lengend Snippet: Schematic drawing of the chr13q22.1 pancreatic cancer risk locus. The top panel shows recombination rate (cM/Mb) in the CEU population relative to the risk locus (top risk SNPs, shown by red vertical bars), the nongenic region and the surrounding genes: MZT1, BORA, DIS3, PIBF1, KLF5 and KLF12. The lower panels show three sub-regions of the risk locus, each containing one or more of the eight highly correlated variants strongly associated with risk of pancreatic cancer and their imputed P value rank. The reported GWAS SNP is rs9543325. For each of the eight strongly-associated variants, layered H3K4Me1, H3K4Me3 and H3K27Ac chromatin immunoprecipitation (ChIP-seq), DNAse I hypersensitivity sequencing (DNase), and transcription factor ChIP-seq (TF ChIP-seq) data from the ENCODE project are shown, as displayed by the UCSC Genome Browser.

Article Snippet: The Pfaffl method ( 51 ) was used to calculate expression values of BORA , DIS3 , PIBF1 , KLF5 and KLF12 (#Hs00227229_m1, #Hs00209014_m1, #Hs00197131_m1, #Hs00156145_m1, #Hs00255460_m1, respectively, Applied Biosystems) by normalizing to the geometric mean of three housekeeping genes ( B2M #Hs00187842_m1, GAPDH #Hs99999905_m1 and PPIA #Hs99999904_m1, Applied Biosystems) and modified by a plate scaling factor based on identical cDNA standards on each plate.

Techniques: Chromatin Immunoprecipitation, ChIP-sequencing, Sequencing

Journal: Human Molecular Genetics

Article Title: Functional characterization of a chr13q22.1 pancreatic cancer risk locus reveals long-range interaction and allele-specific effects on DIS3 expression

doi: 10.1093/hmg/ddw300

Figure Lengend Snippet: eQTL results for candidate functional variants and cis-genes

Article Snippet: The Pfaffl method ( 51 ) was used to calculate expression values of BORA , DIS3 , PIBF1 , KLF5 and KLF12 (#Hs00227229_m1, #Hs00209014_m1, #Hs00197131_m1, #Hs00156145_m1, #Hs00255460_m1, respectively, Applied Biosystems) by normalizing to the geometric mean of three housekeeping genes ( B2M #Hs00187842_m1, GAPDH #Hs99999905_m1 and PPIA #Hs99999904_m1, Applied Biosystems) and modified by a plate scaling factor based on identical cDNA standards on each plate.

Techniques: Functional Assay

Journal: Molecular Therapy

Article Title: LINC00470 Coordinates the Epigenetic Regulation of ELFN2 to Distract GBM Cell Autophagy

doi: 10.1016/j.ymthe.2018.06.019

Figure Lengend Snippet: ELFN2 Binds to AurkA and eIF2α and Upregulates eIF2α by Regulating the Kinase Activity of AurkA (A) Representative confocal and immunofluorescence images showing the co-localization of ELFN2 (red) and AurkA (green) in HEK293 cells. (B) coIP analysis showing the interaction between ELFN2 and AurkA in HEK293 and U251 cells. (C) coIP analysis showing the interaction between ELFN2 and AurkA domains in HEK293 cells. (D) Representative confocal and immunofluorescence images showing the co-localization of ELFN2 (red) and eIF2α (green) in HEK293 cells. (E) coIP analysis showing the interaction between ELFN2 and eIF2α in HEK293 cells. (F) Representative confocal and immunofluorescence images showing the co-localization of AurkA (red) and eIF2α (green) in HEK293 cells. (G) coIP analysis showing the interaction between AurkA and eIF2α in HEK293 cells. (H) GST pull-down assays showed that the reg2 domain of AurkA pulled down ELFN2. (I) GST pull-down assays showed that the reg1 and reg2 domains of AurkA pulled down eIF2α. (J) qRT-PCR was performed to detect the expression of eIF2α in GBM cells after the ELFN2 knockdown. The data are presented as the means ± SEM of three independent experiments. *p < 0.05. (K) Western blotting was performed to detect the levels of AurkA and p-AurkA in GBM cells transfected with si-ELFN2. (L) qRT-PCR was performed to detect the expression of eIF2α in GBM cells after ELFN2 knockdown and AurkA overexpression. The data are presented as the means ± SEM of three independent experiments. **p < 0.01. (M) Western blotting was performed to detect the level of autophagy markers in ELFN2-overexpessing GBM cells after AurkA or eIF2α knockdown.

Article Snippet: The reagents, chemicals, and antibodies used in this study are as follows: LC3 (Cell Signaling Technology, 12741), Beclin-1 (Cell Signaling Technology, 3495), Atg5 (Cell Signaling Technology, 12994), Atg12 (Cell Signaling Technology, 4180), Atg7 (Cell Signaling Technology, 8558), Atg3 (Cell Signaling Technology, 3415), GFP (Proteintech, 50430-2-AP), ELFN2 (Sigma, HPA00781), phospho-AurkA (Cell Signaling Technology), AurkA (Cell Signaling Technology, 14475), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Proteintech, 60004-1-Ig), FLAG (Sigma-Aldrich, F3165), and rapamycin (MedChemExpress, HY-10219).

Techniques: Activity Assay, Immunofluorescence, Quantitative RT-PCR, Expressing, Western Blot, Transfection, Over Expression

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Aurora kinase A controls meiosis I progression in mouse oocytes

doi:

Figure Lengend Snippet: Protein expression and subcellular localization of AURKA during meiotic maturation. (A) Immunoblot blot analysis of AURKA in cumulus-free oocytes (200 oocytes per lane) cultured in vitro to various stages—GV (0 h), GVBD (1 h), MI (7 h), MII (18 h). The amount of AURKA protein increased slightly around GVBD. (B) Quantification of immunoblots. The experiment was performed three times and the data are expressed as mean ±SEM. Statistical differences (p < 0.05) in comparison to GV-stage are marked (*) (C) Phase-contrast and fluorescence microscopy images of oocytes after co-injection of Gfp-Aurka mRNA (2–5 pl of 200 ng/μl) and mCherry-H2B (2–5 pl of 25 ng/μl) into GV-stage. Total AURKA was mainly present at MTOC at GV-stage oocytes, at MI and MII AURKA was mainly associated with spindle with a clear concentration on the spindle poles and cytoplasmic MTOCs (arrowheads).

Article Snippet: For microtubule visualization using immunocytochemistry, mouse monoclonal anti-acetylated α-tubulin antibody (Sigma, T7451) was used. pT288 AURKA was detected by immunocytochemistry using a rabbit anti-phosphorylated T288 AURKA antibody (Novus Biological, NB100-2371) or an anti-phospho-T288 monoclonal AURKA antibody 42 for double-staining with phospho-PKB. pS473 PKB was detected by a rabbit polyclonal antibody pAkt-Ser473 (Santa Cruz, SC-7985) and pT308 PKB by a rabbit polyclonal antibody anti-Akt/PKB [pT308] (BioSource, 44-6026).

Techniques: Expressing, Western Blot, Cell Culture, In Vitro, Comparison, Fluorescence, Microscopy, Injection, Concentration Assay

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Aurora kinase A controls meiosis I progression in mouse oocytes

doi:

Figure Lengend Snippet: PI3K-PKB and CDK1 independent AURKA activation precedes resumption of meiosis. Localization of pT288 AURKA (active form) during meiotic maturation (A). MTOC-associated AURKA is phosphorylated on T288 before GVBD (bGVBD), then during prometaphase I (PMI) and metaphase I (MI) pT288 AURKA remains associated with MTOCs. Green—pT288 AURKA, red—pericentrin (MTOCs). (B) Inhibition of CDK1 activity does not prevent activation of AURKA and multiplication of MTOCs. Oocytes, cultured for 90 min in the presence of Roscovitine, showed phosphorylation of AURKA on T288 at amplified MTOCs or within the nucleus. (C) Ability of LY-294002 or SH-6 oocytes to resume meiosis. The experiment was performed four times and around 200 oocytes were counted for each group. Error bars show confidence intervals. Statistical differences (p < 0.05) in comparison to control are marked (*) (D) PI3K-PKB signaling pathway is not involved in AURKA activation. Oocytes were cultured in medium supplemented with LY-294002 or SH-6 for 90 min and then GV-stage oocytes were used for imunofluorescence staining. Inhibition of PI3K or PKB did not block phosphorylation of AURKA, multiplication of MTOC and induced presence of pT288 AURKA within the nucleus.

Article Snippet: For microtubule visualization using immunocytochemistry, mouse monoclonal anti-acetylated α-tubulin antibody (Sigma, T7451) was used. pT288 AURKA was detected by immunocytochemistry using a rabbit anti-phosphorylated T288 AURKA antibody (Novus Biological, NB100-2371) or an anti-phospho-T288 monoclonal AURKA antibody 42 for double-staining with phospho-PKB. pS473 PKB was detected by a rabbit polyclonal antibody pAkt-Ser473 (Santa Cruz, SC-7985) and pT308 PKB by a rabbit polyclonal antibody anti-Akt/PKB [pT308] (BioSource, 44-6026).

Techniques: Activation Assay, Inhibition, Activity Assay, Cell Culture, Phospho-proteomics, Amplification, Comparison, Control, Staining, Blocking Assay

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Aurora kinase A controls meiosis I progression in mouse oocytes

doi:

Figure Lengend Snippet: Overexpression of AURKA triggers MTOCs multiplication in GV-arrested oocytes. Gfp-Aurka. mRNA was microinjected into GV-blocked oocytes (2–5 pl of 250 ng/μl); Egfp mRNA was used as a control. (A) Exogenous AURKA caused multiplication of MTOCs in the presence of an intact GV after overnight incubation. GFP-AURKA was located on all MTOCs, (B) but 2 h after injection was non-phosphorylated. Overnight incubation led to phosphorylation of exogenous GFP-AURKA on T288. Pericentrin was used as a MTOC marker. (C) Endogenous AURKA is not phosphorylated during overnight culture in IBMX-containing medium (D) Activity of CDK1 (arbitrary units) was assayed on extracts from 10 oocytes per sample. The experiment was done 3 times and activity is expressed as mean ± SEM. Statistical difference (p < 0.05) in comparisons to GV-stage control is marked (*).

Article Snippet: For microtubule visualization using immunocytochemistry, mouse monoclonal anti-acetylated α-tubulin antibody (Sigma, T7451) was used. pT288 AURKA was detected by immunocytochemistry using a rabbit anti-phosphorylated T288 AURKA antibody (Novus Biological, NB100-2371) or an anti-phospho-T288 monoclonal AURKA antibody 42 for double-staining with phospho-PKB. pS473 PKB was detected by a rabbit polyclonal antibody pAkt-Ser473 (Santa Cruz, SC-7985) and pT308 PKB by a rabbit polyclonal antibody anti-Akt/PKB [pT308] (BioSource, 44-6026).

Techniques: Over Expression, Control, Incubation, Injection, Phospho-proteomics, Marker, Activity Assay

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Aurora kinase A controls meiosis I progression in mouse oocytes

doi:

Figure Lengend Snippet: MI spindle defects induced by AURKA overexpression. (A) Aurka mRNA (2–5 pl, 350 ng/ul) injected oocytes, cultured for 18 h, were evaluated. Overexpression of AURKA blocks oocytes in PMI/MI. Altogether 100 GFP (control) and 99 GFP-AURKA oocytes were evaluated in three independent experiments. Error bars show confidence intervals. Statistical differences (p < 0.05) in individual cell cycle stages (MI and MII) are marked (*), difference in GV stage is not significant (B) Morphological defects such as formation of abnormally long spindle, absence of congression of tetrads or formation of unipolar (arrow) and multipolar spindles (arrowheads) were analyzed by immunocytochemistry. The 83 injected oocytes from three independent experiments were evaluated and morphologically analyzed. Red—acetylated α-tubulin, blue—DAPI.

Article Snippet: For microtubule visualization using immunocytochemistry, mouse monoclonal anti-acetylated α-tubulin antibody (Sigma, T7451) was used. pT288 AURKA was detected by immunocytochemistry using a rabbit anti-phosphorylated T288 AURKA antibody (Novus Biological, NB100-2371) or an anti-phospho-T288 monoclonal AURKA antibody 42 for double-staining with phospho-PKB. pS473 PKB was detected by a rabbit polyclonal antibody pAkt-Ser473 (Santa Cruz, SC-7985) and pT308 PKB by a rabbit polyclonal antibody anti-Akt/PKB [pT308] (BioSource, 44-6026).

Techniques: Over Expression, Injection, Cell Culture, Control, Immunocytochemistry

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Aurora kinase A controls meiosis I progression in mouse oocytes

doi:

Figure Lengend Snippet: Downregulation of AURKA leads to incorrect spindle assembly and PMI/MI arrest. Oocytes injected with Aurka dsRNA or with Egfp dsRNA as a control were cultured for 24 h in IBMX-supplemented medium. (A) Aurka mRNA level in relative arbitrary units after RNAi mediated knockdown in GV-arrested oocytes. Total RNA was subsequently isolated and used for real-time PCR to quantify the level of Aurka mRNA. Egfp was used as an external standard. The decrease in the amount of Aurka mRNA was significant (p < 0.05). Data are expressed as the mean ± SEM. (B) Meiotic maturation of dsRNA injected oocytes. Oocytes after microinjection of Aurka dsRNA are arrested PMI/MI, when evaluated 18 h after IBMX release. 109 GFP RNAi and 94 AURKA RNAi oocytes were analyzed in three experiments. Statistical differences (p < 0.05) in individual cell cycle stages (GV, MI and MII) are marked (*) (C) Phenotypes of AURKA knockdown in mouse oocytes. Representative images of three experiments (approx. 100 oocytes) showing contol MI phase spindle of oocytes injected with Egfp (left) and oocytes injected with Aurka dsRNA (right) at the time 7 h after their transfer to IBMX-free medium. Red—acetylated α-tubulin, blue—DAPI for DNA staining.

Article Snippet: For microtubule visualization using immunocytochemistry, mouse monoclonal anti-acetylated α-tubulin antibody (Sigma, T7451) was used. pT288 AURKA was detected by immunocytochemistry using a rabbit anti-phosphorylated T288 AURKA antibody (Novus Biological, NB100-2371) or an anti-phospho-T288 monoclonal AURKA antibody 42 for double-staining with phospho-PKB. pS473 PKB was detected by a rabbit polyclonal antibody pAkt-Ser473 (Santa Cruz, SC-7985) and pT308 PKB by a rabbit polyclonal antibody anti-Akt/PKB [pT308] (BioSource, 44-6026).

Techniques: Injection, Control, Cell Culture, Knockdown, Isolation, Real-time Polymerase Chain Reaction, Microinjection, Staining