Journal: PLoS ONE

Article Title: The HDAC Inhibitor LBH589 Induces ERK-Dependent Prometaphase Arrest in Prostate Cancer via HDAC6 Inactivation and Down-Regulation

doi: 10.1371/journal.pone.0073401

Figure Lengend Snippet: ( A – B ) Immuno-staining of LNCaP and PC-3. Both cells were treated 75 nM LBH589 for 24 h. Immuno-staining ( A ) with Cdc2 and Cdc25C antibodies and ( B ) of γ-tubulin (Green) and DAPI (Blue) in vehicle control (left) and LBH589 treatment (right) in LNCaP. ( C – D ) MEK inhibitor attenuated LBH589-induced prometaphase arrest in LNCaP. The cells were pre-treated 30 with UO126 for 30 minutes and sequentially treated with LBH589 for 24 h. ( C ) The cell cycles were analyzed by PI-staining and flow cytometry. ( D ) The metaphase cells were counted by DAPI staining presented as condensate chromatin.

Article Snippet: The antibodies included HDAC1 (#2062), c-Raf (#9422), Akt (#9272), Pi-Akt (#4058), Pi-Cdc25C (Ser216) (#9528), Pi-c-Raf (S259) (# 9421), Pi-c-Raf (S338) (#9427), and Pi-Cdc2 (Tyr15) (#9111) purchased from Cell Signaling Technology (Beverly, MA, USA); HDAC6 (sc-28386), Pi-ERK(sc-7383), ERK (sc-94), 14-3-3ζ (sc-1019), and Cdc2 (sc-54) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Aurora A (#07-648), H3-Ac (#06-599), H4-Ac (#06-598), p21 (#05-345), and PP2A (#05-421) from Millipore (Lake Placid, NY, USA); GAPDH and actin (Sigma-Aldrich); Aurora B (#1788-1), Cdc25C (#1302-1), and PP1 (#1950-1) from Epitomics (Burlingame, CA); and HDAC3 (ab32369) from Abcam (Cambridge, UK).

Techniques: Immunostaining, Staining, Flow Cytometry

Journal: Biology Direct

Article Title: 14-3-3 theta binding to cell cycle regulatory factors is enhanced by HIV-1 Vpr

doi: 10.1186/1745-6150-3-17

Figure Lengend Snippet: Increased Cdk1, Cdc25C and CyclinB1 association with 14-3-3 θ but stable nucleocytoplasmic distribution during HIV- and Vpr-induced G 2 ,M arrest. Jurkat cells were infected as in Fig. 1 with RT- NL4-3 e-n-GFP virions either with (Vpr v ) or without (Δ) hVpr supplied in trans or with RT+ NL4-3 e-n-GFP (HIV; MOI 2). (A) Cell lysates were harvested two days post-infection for immunoprecipitation with 14-3-3 θ and immunoblotting for CyclinB1, Cdc25C-P.S216, Cdc25C, Cdk1-P.Y15, Cdk1, 14-3-3 θ, and Vpr as indicated. The 14-3-3 θ signal in the IP does not reflect poor immunoprecipitation of 14-3-3 θ but rather the result of membrane stripping prior to 14-3-3 θ blotting. (B) DNA content analysis (y-axis) is shown in flow cytometric dot plots against GFP (x-axis) on the right and as a histogram in the inset for the samples in (A). Note that the y-axis of the parent graph becomes the x-axis of the inset graph. The quadrant gate demarcates approximate G1 (lower) and S/G 2 ,M (upper) populations and the percentage of cells in each relevant quadrant is indicated. The DNA histogram profile analysis was separated into GFP-positive (+) and negative (-) populations by the x-axis gate for the HIV-infected culture; as expected the infected cells (+) show G 2 arrest, but the uninfected cells (-) are mostly G1. (C) G 2 ,M cell cycle arrest caused by Vpr v and HIV infection does not alter the cytoplasmic and nuclear distribution of 14-3-3 θ, Cdc25C, Cdk1, and CyclinB1. Jurkat T cells shown in (A-B) that were infected with NL4-3 e-n-GFP RT- Δ Vpr (Δ), RT- wt Vpr (Vpr v ), or NL4-3 e-n-GFP RT+ (HIV) for two days were lysed and biochemically separated into cytoplasmic and nuclear fractions. Lysate fractions were blotted as in (A) (the lower panel of Vpr blot represents a longer exposure in which Vpr v is more apparent), with the addition of probes for HIV-1 Vif, Poly(ADP-ribose) polymerase (PARP) as a nuclear marker, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a cytoplasmic loading control. Cell cycle profiles and GFP expression are shown in (B). (D) Viral lysates (20 μg) of RT- NL4-3 e-n-GFP virions with (+) or without (-) Vpr were western blotted for CyclinB1, Cdk1, p24, and Vpr as indicated.

Article Snippet: Antibodies used with corresponding catalog numbers are as follows: β-actin (Sigma; A5441), Cdc2 (Santa Cruz; sc-54), Phospho-Tyr15-Cdc2 (Cell Signaling; 9111), Cdc25C (BD Pharmingen; 550922), Cdc25C (Santa Cruz; sc-327), Cdc25C (Calbiochem; CC26; EMSA), Phospho-Ser216-Cdc25C (Cell Signaling; 4901), Centrin (Sigma; C7736), CyclinB1 (Santa Cruz; sc-752 or sc-245), GAPDH (Abcam; ab9484), PARP (BD Transduction Laboratories; P76420), importin β (Sigma; I2534) PKA (Santa Cruz; sc-903), Plk1 (Upstate; 05–844), γ-Tubulin (Sigma; T3559); HIV-1 Vif (NIH ARRRP; 6459), HIV-1 Vpr (gift of K. Strebel, NIAID), 14-3-3 β (Santa Cruz; sc-25276), 14-3-3 θ (Santa Cruz; sc-732), 14-3-3 γ (Santa Cruz; sc-731c), 14-3-3 ζ (Santa Cruz; sc-1019; cross-reacts with β and σ isoforms).

Techniques: Infection, Immunoprecipitation, Western Blot, Stripping Membranes, Marker, Expressing

Journal: Biology Direct

Article Title: 14-3-3 theta binding to cell cycle regulatory factors is enhanced by HIV-1 Vpr

doi: 10.1186/1745-6150-3-17

Figure Lengend Snippet: Mutant Vpr fails to stimulate the association of CyclinB1, Cdk1, and Plk1 with 14-3-3 θ but still binds 14-3-3 θ itself. (A) Jurkat cells were infected as in Fig. 2 with RT- NL4-3 e-n-GFP virions (Vpr v ) lacking Vpr (Δ) or containing either wild-type (wt), R80A mutant (80A), or I70S mutant (70S) Vpr. Cells lysates were harvested two days post-infection. for immunoprecipitation with 14-3-3 θ antibody (IP: 14-3-3 θ) and the lysates (input) and IP were blotted as indicated with antibodies recognizing Plk1, CyclinB1, Cdk7, Cdk1, 14-3-3 θ, and Vpr. (B) DNA content analysis of the samples in (A), top row (Vpr v ), and of aphidicolin-synchronized Jurkat cells released from the G1 block for the indicated number of hours (bottom row; sync.). (C) Immunoprecipitation and western blot were performed as in (A) of lysates from the cell cycle synchronized cells shown in (B, bottom row). (D) Jurkat cells were infected with NL4-3 e-n-GFP (RT+) derivatives containing either wild-type Vpr and Vif (lane 1), Vpr but no Vif (lane 2), neither Vpr nor Vif (lane 3), or R80A mutant Vpr and no Vif (lane 4). Two days post-infection cells were lysed, immunoprecipitated, and immunoblotted as in (A) (top). Flow cytometric DNA content analysis was performed at the time of harvest and shown for the GFP+ (HIV-infected) population of each sample (bottom, numbering corresponds to lane numbers of blots). GFP expression is shown as an inset with the percentage of cells in the GFP-positive gate indicated within the plot.

Article Snippet: Antibodies used with corresponding catalog numbers are as follows: β-actin (Sigma; A5441), Cdc2 (Santa Cruz; sc-54), Phospho-Tyr15-Cdc2 (Cell Signaling; 9111), Cdc25C (BD Pharmingen; 550922), Cdc25C (Santa Cruz; sc-327), Cdc25C (Calbiochem; CC26; EMSA), Phospho-Ser216-Cdc25C (Cell Signaling; 4901), Centrin (Sigma; C7736), CyclinB1 (Santa Cruz; sc-752 or sc-245), GAPDH (Abcam; ab9484), PARP (BD Transduction Laboratories; P76420), importin β (Sigma; I2534) PKA (Santa Cruz; sc-903), Plk1 (Upstate; 05–844), γ-Tubulin (Sigma; T3559); HIV-1 Vif (NIH ARRRP; 6459), HIV-1 Vpr (gift of K. Strebel, NIAID), 14-3-3 β (Santa Cruz; sc-25276), 14-3-3 θ (Santa Cruz; sc-732), 14-3-3 γ (Santa Cruz; sc-731c), 14-3-3 ζ (Santa Cruz; sc-1019; cross-reacts with β and σ isoforms).

Techniques: Mutagenesis, Infection, Immunoprecipitation, Blocking Assay, Western Blot, Expressing

Journal: Biology Direct

Article Title: 14-3-3 theta binding to cell cycle regulatory factors is enhanced by HIV-1 Vpr

doi: 10.1186/1745-6150-3-17

Figure Lengend Snippet: Reduced 14-3-3 θ association with centrosomal proteins, centrin and Plk1, during HIV-1 infection-induced G 2 ,M arrest. (A) Jurkat cells were mock-infected (-) or infected with NL4-3 e-n-GFP derivatives encoding wild-type Vif and Vpr (wt), deleted Vpr (r-), deleted Vif (f-), or double deletion of Vpr and Vif (fr-) at an MOI of 1.5. Lysates were harvested two days post-infection and immunoprecipitated as in Fig. 2 (IP: 14-3-3 θ). Whole cell lysates (input) and IP samples were blotted for Plk1, 14-3-3 θ, centrin, Vif, and Vpr. There appeared to be poor transfer of centrin protein at the left edge of the gel (input lanes). The reduced band intensity for this sample does not reflect decreased centrin abundance as it was well-represented in the IP and similar experiments showed no changes in centrin expression upon HIV-1 infection. (B) DNA content analysis of the samples in (A) by propidium iodide staining. HIV-infected samples were pre-gated on GFP+ cells for DNA analysis. (C) Viability (large plot) and GFP expression by viable cells (inset) for samples in (A) and (B) were measured by flow cytometric detection of propidium iodide (PI) negative, large (high forward scatter) cells and GFP fluorescence (inset histogram), respectively, at the time lysates were harvested. Plots correspond to samples directly above in (B). The gates demarcate viable and GFP-positive cell populations and the percentage of cells within each gate is indicated. (D) Jurkat cells were mock-infected (m), infected with Vpr v as in (A), or infected with NL4-3 e-n-GFP (HIV) and harvested after 40 hours for immunoprecipitation with 14-3-3 θ and immunoblotting with importin β, CyclinB1, 14-3-3 θ, and Vpr. (E) DNA content analysis performed as in (B) is shown for the samples in (D). The inset histogram depicts the percentage of GFP+ cells expressing the NL4-3 e-n-GFP provirus. The DNA analysis for the HIV-infected sample was performed on the gated GFP population indicated. (F) Jurkat cells were mock-infected (m), infected with RT- NL4-3 e-n-GFP virions (Vpr v ) to deliver Vpr protein, or treated with adriamycin (adr). Cell lysates (input) were harvested after 40 hours for immunoprecipitation with importin β (IP) and immunoblotting with importin β, CyclinB1, Plk1, Cdk1, and Vpr as indicated. (G) Cell cycle analysis is shown for the samples in (F) at the time of lysis as measured by propidium iodide DNA staining as in (B).

Article Snippet: Antibodies used with corresponding catalog numbers are as follows: β-actin (Sigma; A5441), Cdc2 (Santa Cruz; sc-54), Phospho-Tyr15-Cdc2 (Cell Signaling; 9111), Cdc25C (BD Pharmingen; 550922), Cdc25C (Santa Cruz; sc-327), Cdc25C (Calbiochem; CC26; EMSA), Phospho-Ser216-Cdc25C (Cell Signaling; 4901), Centrin (Sigma; C7736), CyclinB1 (Santa Cruz; sc-752 or sc-245), GAPDH (Abcam; ab9484), PARP (BD Transduction Laboratories; P76420), importin β (Sigma; I2534) PKA (Santa Cruz; sc-903), Plk1 (Upstate; 05–844), γ-Tubulin (Sigma; T3559); HIV-1 Vif (NIH ARRRP; 6459), HIV-1 Vpr (gift of K. Strebel, NIAID), 14-3-3 β (Santa Cruz; sc-25276), 14-3-3 θ (Santa Cruz; sc-732), 14-3-3 γ (Santa Cruz; sc-731c), 14-3-3 ζ (Santa Cruz; sc-1019; cross-reacts with β and σ isoforms).

Techniques: Infection, Immunoprecipitation, Expressing, Staining, Fluorescence, Western Blot, Cell Cycle Assay, Lysis

Journal: Biology Direct

Article Title: 14-3-3 theta binding to cell cycle regulatory factors is enhanced by HIV-1 Vpr

doi: 10.1186/1745-6150-3-17

Figure Lengend Snippet: Cell cycle regulatory protein binding to 14-3-3 θ is also enhanced during G 2 ,M arrest induced by adriamycin. Jurkat T cells were treated with adriamycin (Adr; 0.2 μg/ml) for 24, 48, or 72 hours (h) or untreated (0) and examined for 14-3-3 θ co-immunoprecipitating (IP) proteins. (A) Western blot analysis of Plk1, CyclinB1, Cdk1, 14-3-3 θ and centrin in whole cell lysates before IP (input) and after 14-3-3 θ IP (IP: 14-3-3 θ) at the time of adriamycin treatment indicated. These data are representative of three independent experiments. Although the amount of 14-3-3 θ appears less in the untreated (0) time point of the IP, this was not a reproducible finding. (B) Viability of the samples used in the IP in (A) determined by flow cytometry detection of propidium iodide (PI) exclusion and large size (high forward scatter). The percentage of viable cells is indicated. Flow cytometric histograms of the DNA content for each sample determined by propidium iodide DNA staining (x-axis) are shown as an inset.

Article Snippet: Antibodies used with corresponding catalog numbers are as follows: β-actin (Sigma; A5441), Cdc2 (Santa Cruz; sc-54), Phospho-Tyr15-Cdc2 (Cell Signaling; 9111), Cdc25C (BD Pharmingen; 550922), Cdc25C (Santa Cruz; sc-327), Cdc25C (Calbiochem; CC26; EMSA), Phospho-Ser216-Cdc25C (Cell Signaling; 4901), Centrin (Sigma; C7736), CyclinB1 (Santa Cruz; sc-752 or sc-245), GAPDH (Abcam; ab9484), PARP (BD Transduction Laboratories; P76420), importin β (Sigma; I2534) PKA (Santa Cruz; sc-903), Plk1 (Upstate; 05–844), γ-Tubulin (Sigma; T3559); HIV-1 Vif (NIH ARRRP; 6459), HIV-1 Vpr (gift of K. Strebel, NIAID), 14-3-3 β (Santa Cruz; sc-25276), 14-3-3 θ (Santa Cruz; sc-732), 14-3-3 γ (Santa Cruz; sc-731c), 14-3-3 ζ (Santa Cruz; sc-1019; cross-reacts with β and σ isoforms).

Techniques: Protein Binding, Western Blot, Flow Cytometry, Staining

Journal: Biology Direct

Article Title: 14-3-3 theta binding to cell cycle regulatory factors is enhanced by HIV-1 Vpr

doi: 10.1186/1745-6150-3-17

Figure Lengend Snippet: Cell cycle regulatory proteins reside in the centrosome during G 2 ,M arrest induced by HIV-1 infection and adriamycin. (A) Western blot analysis of centrosomes isolated from Jurkat cells infected with NL4-3 e-n-GFP (HIV; MOI of 2) for two days. Centrosomes were isolated by discontinuous sucrose gradient and fractions were collected and separated by SDS-PAGE and western blotted for Plk1, CyclinB1, γ-tubulin, Cdk1, 14-3-3 θ, centrin, Vif, and Vpr. Lanes 1–6 represent fractions from the bottom of the gradient upward, with centrosomes most abundant in lane 3. Whole cell lysates were run in lane 8 and volume from the top of the gradient equivalent to that used for each fraction was run in lane 7 to demonstrate sedimentation of centrosomal proteins through the gradient. "L" indicates a light exposure and "D" indicates a darker exposure of the chemilumigraph of the middle part of the gel. (B) Viability (top) and DNA content analysis (bottom) by flow cytometry for the culture in (A; HIV (+)) and untreated Jurkats without (-) HIV infection. The percentage of viable cells was determined by propidium iodide (PI) exclusion and high forward scatter and is indicated in the lower right corner. Infection efficiency was measured by GFP expression (inset; gated population) and the percentage is indicated. DNA content was measured by flow cytometric detection of DNA stained with propidium iodide. The GFP-positive population was analyzed for the HIV-infected culture. (C) Jurkat T cells were treated with adriamycin (adr; 0.2 μg/ml; right panel) for two days or grown asynchronously (left panel) and centrosomes were isolated by discontinuous sucrose gradients as in (A). Centrosomes were most abundant in lanes 2–3 (untreated cells) or lanes 3–4 (adriamycin treated cells). Lanes were as described in panel A. (D) Cell cycle flow cytometric analysis of Jurkat cells in (C).

Article Snippet: Antibodies used with corresponding catalog numbers are as follows: β-actin (Sigma; A5441), Cdc2 (Santa Cruz; sc-54), Phospho-Tyr15-Cdc2 (Cell Signaling; 9111), Cdc25C (BD Pharmingen; 550922), Cdc25C (Santa Cruz; sc-327), Cdc25C (Calbiochem; CC26; EMSA), Phospho-Ser216-Cdc25C (Cell Signaling; 4901), Centrin (Sigma; C7736), CyclinB1 (Santa Cruz; sc-752 or sc-245), GAPDH (Abcam; ab9484), PARP (BD Transduction Laboratories; P76420), importin β (Sigma; I2534) PKA (Santa Cruz; sc-903), Plk1 (Upstate; 05–844), γ-Tubulin (Sigma; T3559); HIV-1 Vif (NIH ARRRP; 6459), HIV-1 Vpr (gift of K. Strebel, NIAID), 14-3-3 β (Santa Cruz; sc-25276), 14-3-3 θ (Santa Cruz; sc-732), 14-3-3 γ (Santa Cruz; sc-731c), 14-3-3 ζ (Santa Cruz; sc-1019; cross-reacts with β and σ isoforms).

Techniques: Infection, Western Blot, Isolation, SDS Page, Sedimentation, Flow Cytometry, Expressing, Staining

Journal: PLoS ONE

Article Title: Efficient Induction of Apoptosis by Wee1 Kinase Inhibition in Hepatocellular Carcinoma Cells

doi: 10.1371/journal.pone.0100495

Figure Lengend Snippet: A, FACS analyses showed that the sub-G1 phase HuH7 cell population increased from approximately 0.7% to 7.7%, and cells in the G1 phase increased from approximately 69% to 87% 48 h after TGF-β1 treatment. Data represent the means ± S.D. of three experiments. * indicates significant differences between each group (P<0.05). B, Cdc2 kinase activity was determined based on the level of phosphorylated histone H1 using histone H1 as a substrate. Cdc2 was activated 48 h after TGF-β1 treatment in HuH7 cells. However, cdc2 was not activated in HuH7R cells, which were isolated as an apoptosis-resistant clone from TGF-β1-treated HuH7 cells. Roscovitine (Ros)-pretreated HuH7 cells did not show cdc2 activation. A representative image from three independent experiments is shown. C, After TGF-β1 treatment (48 h), we observed cdc2 Tyr15 dephosphorylation in association with Wee1 kinase down-regulation in apoptotic cells. Pretreatment with 20 µM roscovitine completely abolished apoptosis and restored Wee1 kinase expression. TGF-β1 treatment induced G1 cell cycle arrest in HuH7R cells; however, Wee1 kinase expression and non-phosphorylated cdc2 Tyr 15 were similar to those of roscovitine-pretreated HuH7 cells. A representative image of three experiments is shown. D, Wee1 down-regulation and cdc2 Tyr15 dephosphorylation commenced approximately 24 h after TGF-β1 treatment, which was similar to thea initiation of apoptosis. A representative Western blot image is shown in the upper panels. The results in the lower graphs represent the means ± S.D. of three experiments. * indicates significant differences between each group (P<0.05).

Article Snippet: Immunoblots were prepared as described previously and probed with anti-Wee1, anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), or anti-cdc2-phospho-Tyr15 (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Activity Assay, Isolation, Activation Assay, De-Phosphorylation Assay, Expressing, Western Blot

Journal: PLoS ONE

Article Title: Overexpression of LIM and SH3 Protein 1 Leading to Accelerated G2/M Phase Transition Contributes to Enhanced Tumourigenesis in Oral Cancer

doi: 10.1371/journal.pone.0083187

Figure Lengend Snippet: (A) Flow cytometric determination of DNA content was analyzed using the Accuri C6 Flow Cytometer. shLASP-1-transfected HSC-3 and Ca9-22 cells show significant (* P <0.025; Mann-Whitney U test with Bonferroni correction) G2 phase accumulation as opposed to the shMock-transfected cells. (B) Immunoblotting analysis shows down-regulation of cyclin A and cyclin B and up-regulation of phospho-cdc2 in the knockdown cells.

Article Snippet: Protein extracts were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis in 10% gel and transferred to nitrocellulose membranes before incubation overnight with primary antibodies against LASP-1 (Applied Biological Materials, Richmond, BC, Canada), cyclin A, cyclin B, or phospho-cdc2 (Tyr15) (Cell Signaling Technology, Danvers, MA) at 4°C.

Techniques: Flow Cytometry, Transfection, MANN-WHITNEY, Western Blot