Journal: bioRxiv

Article Title: Suv420 enrichment at the centromere limits Aurora B localization and function

doi: 10.1101/2021.06.14.448441

Figure Lengend Snippet: A) Quantification of anaphase defects showing that cells induced to express cen-Suv39 or cen-Suv420 exhibit an increase in anaphase lagging chromosomes following low nanomolar concentrations of Aurora kinase inhibition while their uninduced counterparts do not. Cells both with and without induction of cen-Suv39-GFP or cen-Suv420-GFP are similarly sensitive to inhibition of the mitotic kinase MPS1. (+/- cen-Suv39-GFP induction: p= 0.00003, 0.0003, and 0.844 for Alisertib, Barasertib, and MPS1IN1, respectively; +/-cen-Suv420-GFP: p = 0.003, 0.004, and 0.724 for Alisertib, Barasertib, and MPS1IN1, respectively) Error bars are +/- SD. B & C) Cen-INCENP-mCherry localizes to centromeres in cen-Suv420-GFP expressing cells. D) Expression of cen-INCENP-mCherry does not limit cen-Suv420-GFP localization to centromeres. GFP-staining intensity between ACA peaks was measured for 3 kinetochore pairs in each of 30 metaphase cells per condition, in each of 3 biological replicates. E) expression of cen-INCENP-mCherry reduces the frequency of lagging chromosomes seen in cen-Suv420-GFP expressing anaphase cells. A minimum of 30 anaphase cells were scored per condition, for each of 3 biological replicates. **: p < 0.01, ***: p < 0.001. F) Quantification of area under the dose response curve (AUC) for each drug in cancer cell lines from the TCGA sorted by top and bottom quartile of either Suv39 or Suv420 expression shows that cells with high expression of either Suv39 or Suv420 exhibit increased sensitivity to Aurora kinase inhibition. Each dot represents an individual cancer cell line tested. Open circles indicate the mean of IC50 AUCs for each condition. *: p < 0.00625, **: p < 0.00125, ***: p <0.000125. Error bars are +/- SEM.

Article Snippet: Expression of centromere-targeted, GFP-tagged cen-GFP, cen-Suv39-GFP and cen-Suv420-GFP fusion proteins, and non-targeted Suv420-GFP was achieved by cloning the respective cDNA into Addgene vector CENP-B DBD INCENP GFP (45237, Addgene) at NheI / BamH1.

Techniques: Inhibition, Expressing, Staining

Journal: The Journal of Cell Biology

Article Title: Aurora A kinase phosphorylates Hec1 to regulate metaphase kinetochore–microtubule dynamics

doi: 10.1083/jcb.201707160

Figure Lengend Snippet: Aurora A kinase binds and localizes to INCENP. (A) Volcano plot of quantitative liquid chromatography–tandem mass spectrometry experiments identifying interactors of INCENP. Plotted are the differences in label-free quantification intensity between the INCENP-GFP (right) and control group against the transformed (−log 10 ) p-value of a Student’s t test. The red line indicates the permutation-based false discovery rate (FDR) threshold (0.001) to correct for multiple testing. (B) Western blot analysis of an anti-GFP immunoprecipitation (IP) performed in mitotic cell lysates, using either nocodazole (N) or STLC (S) from HeLa cells expressing either LAP or INCENP-LAP. After SDS-PAGE, the Western blot was probed for GFP and Aurora A kinase (AAK). 10% of input was loaded. (C) Western blot analysis of an anti-GFP immunoprecipitation performed in cell lysates from HEK-293T cells transiently expressing either GFP, full-length (FL) INCENP-GFP, or various truncation mutants of INCENP-GFP (1–820, 1–878, and 1–897). The Western blot was probed for GFP and Aurora A kinase and reprobed for Aurora B kinase (ABK). 10% of input was loaded. (D) Immunofluorescence images of U2OS cells expressing either LacI-mCherry or LacI-mCherry-ΔCEN-INCENP (a version of INCENP lacking the N-terminal 57 residues; ). Cells were immunostained with an Aurora A kinase antibody. Enlarged images show recruitment of Aurora A kinase to the LacI-mCherry-ΔCEN-INCENP spot. Quantification of Aurora A kinase intensity is shown on the right. (E) Immunofluorescence images of U2OS cells expressing VSV-INCENP-GFP under a doxycycline-inducible promoter, either noninduced or induced. Cells were immunostained with GFP and Aurora A kinase antibodies. Enlarged images show colocalization of INCENP-GFP and Aurora A kinase upon overexpression of INCENP-GFP. (F) Line graphs of kinetochore pairs highlighted in E. (G) Immunofluorescence images showing kinetochore localization of pS69 in HeLa cells treated with siRNA to either luciferase (control) or INCENP (siINCENP). Quantification of pS69 and INCENP fluorescence intensity is shown on the right. For pS69 quantification, n = 3 experiments of 15–20 cells; for INCENP quantification, n = 1 experiment of 15–20 cells. Error bars indicate SD. ****, P < 0.0001; unpaired t test. Bars: (main images) 10 µm; (insets) 1 µm.

Article Snippet: Cells were transfected with a LacI-mCherry or LacI-mCherry–ΔCEN-INCENP vector (45236; Addgene; from M. Lampson; ) using Lipofectamine 2000 (Thermo Fisher Scientific).

Techniques: Liquid Chromatography, Mass Spectrometry, Quantitative Proteomics, Control, Transformation Assay, Western Blot, Immunoprecipitation, Expressing, SDS Page, Immunofluorescence, Over Expression, Luciferase, Fluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: Increased Aurora B expression reduces substrate phosphorylation and induces chromosomal instability

doi: 10.3389/fcell.2022.1018161

Figure Lengend Snippet: Expression of GFP-Aurora B in HeLa cells causes a reduction in Aurora B kinase activity and an increase in mitotic defects. Cells were analyzed 48 h after infection with retroviruses expressing GFP-Aurora B or GFP alone. (A) Immunoblot showing endogenous pT232 (active) Aurora B is reduced in HeLa cells overexpressing Aurora B. Tubulin is shown as a loading control. (B) Immunoblot showing reduced phosphorylation of the bona fide Aurora B substrate histone H3 Serine 10 in HeLa cells infected with retroviruses expressing GFP-Aurora. Tubulin is shown as a loading control. (C–H) Quantitative immunofluorescence demonstrating that expression of GFP-Aurora B decreases phosphorylation of histone H3 (pH3), the kinetochore component DSN1, and INCENP in HeLa cells. (C,E,G) Representative images of pH3, pDSN1, and pINCENP, respectively, in HeLa cells expressing GFP and GFP-Aurora B. (D,F,H) Quantification of pH3 S10 (D) , pDSN1 S109 (F) , and pINCENP TSS (H) demonstrating that expression of GFP-Aurora B significantly reduces phosphorylation of these Aurora B substrates in HeLa cells. n > 10 cells from each of 3 independent experiments. (I–P) GFP-Aurora B expression increases the incidence of mitotic defects. (I) Representative image of a misaligned chromosome (arrow). (J) Expression of GFP-Aurora B in HeLa cells causes an increase in misaligned chromosomes in metaphase. n > 50 metaphase cells from 3 independent experiments. (K) Representative image of a lagging chromosome (arrow). (L) Expression GFP-Aurora B in HeLa cells causes an increase in lagging chromosomes. n > 250 anaphase and telophase cells from each of 3 independent experiments. (M) Representative image of a multipolar spindle. (N) Expression of GFP-Aurora B in HeLa cells causes an increase in multipolar spindles. n > 250 mitotic cells from each of 3 independent experiments. (O) Expression of GFP-Aurora B increases the percentage of binucleate HeLa cells. n > 250 cells from each of three independent experiments. (P) Expression of GFP-Aurora B in HeLa cells impairs their ability to arrest in mitosis after 16 h of treatment with the microtubule poison colcemid, consistent with a weakened mitotic checkpoint. n > 250 cells from each of 3 independent experiments. * p < 0.05.

Article Snippet: Aurora B-3xFLAG was immunoprecipitated with M2-agarose slurry (Selleck B23102) and incubated in buffer (200 mM Tris-HCl, pH 7.5, 100 mM MgCl 2 , 1.5M NaCl, 100 mM NaF) with 1 mM DTT, 1 μM cold ATP, 5 μCi [γ- 32 P] ATP, 2.5 μg histone H3 (NEB M2506S) and with or without the IN-box of recombinant INCENP (Signal Chem I30-31H) at 30°C for 30 min and resolved by SDS-PAGE. γ- 32 P incorporation was visualized by Typhoon TRIO imager (GE Healthcare).

Techniques: Expressing, Activity Assay, Infection, Western Blot, Immunofluorescence

Journal: Journal of Biological Chemistry

Article Title: Overexpression of SR-BI by Adenoviral Vector Reverses the Fibrateinduced Hypercholesterolemia of Apolipoprotein E-deficient Mice

doi: 10.1074/jbc.m310892200

Figure Lengend Snippet: FIG. 5. Assessment of apoB levels in the plasma of ciprofibrate-treated apoE/ mice after adenovirus injec- tion. Animals maintained on a chow diet containing 0.05% ciprofibrate for 30 days were injected through a tail vein with ei- ther 5 1010 particles of murine SR-BI recombinant adenovirus () or with 1 1011 particles of lacZ recombinant adeno- virus () as control for the adenoviral in- fection. At the indicated times, equal vol- umes of plasma from five mice per group were pooled and processed as described under “Experimental Procedures.” a, Western blots were performed with rabbit polyclonal antibodies specific for mouse apoB-48 and apoB-100. Western blots of apoB in wild-type and apoE-deficient mice not treated with ciprofibrate are shown for comparison. b, bars show the densitometric analysis (mean S.D. of three separate determinations) of apoB-48 from the injected animals. The apoB-48 levels from the animals injected with Ad.lacZ (open bars) were arbitrarily assigned the value of 100.

Article Snippet: The membranes were blocked for 1 h in TBST (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20) containing 5% nonfat dried milk and then probed overnight either with rabbit polyclonal antibodies (Novus Biologicals, Littleton, CO) against murine SR-BI (1:1500 dilution in TBST) or a monoclonal antibody (clone AC-15, Sigma) against -actin (1:5000 dilution in TBST) (used as protein loading control).

Techniques: Clinical Proteomics, Injection, Recombinant, Virus, Control, Western Blot, Comparison

Journal: Journal of Biological Chemistry

Article Title: Overexpression of SR-BI by Adenoviral Vector Reverses the Fibrateinduced Hypercholesterolemia of Apolipoprotein E-deficient Mice

doi: 10.1074/jbc.m310892200

Figure Lengend Snippet: FIG. 6. Association of [3H]cholesteryl ether-labeled lipoprotein remnants with control, untransfected, and mSR-BI-transfected cells. Transfected ldlA(mSR-BI) and untransfected ldlA cells grown in six-well culture plates were incubated with serum-free medium con- taining 3H-labeled lipoprotein remnants (120 g/well cholesterol). When indicated, purified goat polyclonal antibody raised against SR-BI (Ab) was added to the incubation medium of the transfected cells (4 g/ml IgG). After 4 h of incubation at 37 °C the wells were washed, the cells were harvested, and their associated radioactivity was measured as described under “Experimental Procedures.” Each bar represents the mean S.D. of measurements carried out in cells collected from four separate wells. *, p 0.05 for ldla(mSR-BI) versus ldlA; **, p 0.05 for ldlA(mSR-BI) Ab versus ldlA(mSR-BI).

Article Snippet: The membranes were blocked for 1 h in TBST (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20) containing 5% nonfat dried milk and then probed overnight either with rabbit polyclonal antibodies (Novus Biologicals, Littleton, CO) against murine SR-BI (1:1500 dilution in TBST) or a monoclonal antibody (clone AC-15, Sigma) against -actin (1:5000 dilution in TBST) (used as protein loading control).

Techniques: Labeling, Control, Transfection, Incubation, Purification, Radioactivity