13 research outputs found

    Cytokinesis proceeds to late stages in suspension culture.

    No full text
    <p>GD25 M-cells were incubated in suspension for 1.5 hours and then immuno-stained for aurora B and CEP55 as described in the Method section.</p

    Schematic model for colony growth in suspension and the formation of binucleated cells.

    No full text
    <p>When the G1 checkpoint is suppressed, detached cells progress in the cell cycle and reach the cytokinesis phase. Cells expressing oncogenes (e.g. overactive Ras) complete the cytokinesis and proliferate in agar. Alternatively, cells can finish cytokinesis after renewed ECM-integrin signals by adhesion or FN assembly. FN deposition on the cell surface involves exposure of FN–FN binding sites by mechanical stretching of the protein, which occurs normally between neighboring cells and is dependent on the stiffness of the support. Consequently, it is very inefficient on single cells in suspension. However, the multilobular structure of cytokinesis-blocked cells after two or more rounds of the cell cycle promotes the assembly of secreted FN. The incidence of binucleation is high if cells enter the cell cycle without completing cytokinesis. Black nuclei represent G1 phase; red nuclei represent S phase; rounded morphology of cells represent non-adherent cells.</p

    Analysis of suspension-induced G1 and cytokinesis blocks in FN deficient 4D cells.

    No full text
    <p>(<b>A</b>) Photographs of 4D M-cells grown in suspension for 12 hours and then incubated with 10 µM EdU for 60 min. (<b>B</b>) 4D cells in suspension retained the cytokinesis block. The number of 4D cell-doublets and single cells were counted directly after isolation of M-cells and after 6 hours suspension culture. (<b>C</b>) Cytokinesis-blocked 4D cells in suspension progressed in the cell cycle. 4D M-cells were EdU-labeled for 60 min with 10 µM EdU after 4 hours or 12 hours in suspension culture, and the number of labeled cells was counted. (<b>D</b>) Photograph of a 4D M-cell grown in suspension (methylcellulose) for 48 hours and then trypsinized as described in Materials and methods to reveal the connection between cell bodies. The bars in (B) and (C) show the results from three independent experiments +/- SD.</p

    Adhesion-induced RICTOR-mediated AKT Ser473 phosphorylation promotes cell-survival.

    No full text
    <p>(A) MCF7 cells transfected with RICTOR-directed or non-target siRNA were grown on glass coverslips in complete culture medium for 48 h, then serum starved for 24 h. A TUNEL assay was performed to visualise apoptotic cells and DAPI was used to stain nuclei. Images shown are representative of pictures taken in three independent experiments. (B) Quantification of (A): Five fields were photographed and at least 400 cells were counted from each coverslip and data is presented as fold change in the percentage of apoptotic cells (mean ± s.e.m.; <i>n</i> = 3, ** represents <i>p</i><0.05). (C) MCF7 cells were transfected as described in (A) and after culturing them for 48 h, the cells were trypsinised and 5×10<sup>5</sup> cells were seeded in starvation medium on invasin- or collagen type I-coated glass coverslips. The cells were incubated in starvation medium for 24 h, fixed, stained with DAPI and observed under a fluorescent microscope. Cells with pyknotic nuclei (condensed and brightly blue-stained) were considered as apoptotic cells. The result is presented as fold change (mean ± s.e.m.; <i>n</i> = 2, * represents <i>p</i><0.005 and ** represents <i>p</i><0.05). (D) MCF7 cells were transfected as explained above and subsequently 1×10<sup>5</sup> cells were seeded onto collagen type I-coated dishes in starvation medium. The cells were trypsinised after the indicated time periods and the number of viable cells was counted using the Trypan blue exclusion method. The plot shows fold change in the percentage of viable cells (mean ± s.e.m.; <i>n</i> = 2 and ** represents <i>p</i><0.05) normalised to non-target transfected cells for each time point. The actual numbers of viable cells are given in the table below.</p

    Effects of RICTOR, ILK or PAK knockdown on LPA-induced AKT Ser473 phosphorylation in HeLa and MCF7 cells.

    No full text
    <p>(A) HeLa cells were transfected with RICTOR-directed siRNA or non-target siRNA and then stimulated with LPA (10 µM) for 20 minutes. Cell lysates were subjected to SDS-PAGE (4–15% gradient gel) and LPA-induced AKT Ser473 phosphorylation was determined by western blotting. A representative western blot is shown and the graph below provides quantification of AKT pSer473 (mean ± s.e.m.; <i>n</i> = 3). (B) HeLa cells transfected with ILK-directed siRNA and stimulated by LPA were analysed as described above. The graph below shows quantification of AKT pSer473 (mean ± s.e.m.; <i>n</i> = 2). (C) MCF7 cells transfected as indicated were stimulated with LPA (5 µM) for 5 min and analysed as described above. A representative western blot is presented and the graph below shows quantified levels of AKT pSer473 (mean ± s.e.m.; <i>n</i> = 3, * represents <i>p</i><0.005). (D) MCF7 cells were transfected simultaneously with PAK1 and PAK2 siRNA or with non-target siRNA and then stimulated with LPA (5 µM) for 5 min. A representative western blot is shown. The graph below is a quantification of AKT pSer473 levels (mean ± s.e.m.; <i>n</i> = 3, ** represents <i>p</i><0.05). (E) HeLa cells were treated with increasing concentrations of the PAK inhibitor IPA3 or with DMSO and then stimulated with LPA (10 µM) for 20 min. The graph below is the quantification of AKT pSer473 levels after LPA-stimulation in cells treated with IPA3 (30 µM) normalised to the pSer473 level of this protein in LPA-stimulated cells without the inhibitor (mean ± s.e.m.; <i>n</i> = 3, * represents <i>p</i><0.005).</p

    RICTOR knockdown reduces β1 integrin-induced AKT Ser473 phosphorylation but ILK or PAK knockdown has no effect.

    No full text
    <p>(A) HeLa cells were transfected with siRNA directed against RICTOR and then allowed to adhere to plates coated with invasin (β1 integrin-ligand) or Pluronic (non-adhesive control) for 60 min. Cell lysates were subjected to SDS-PAGE (4–15% gradient gel) followed by western blotting using the different antibodies as indicated. A representative western blot is shown and quantifications of AKT pSer473, FOXO1 pThr24, and BAD pSer136 on Pluronic and invasin are given below (mean ± s.e.m.; <i>n</i> = 3; * represents <i>p</i><0.005 and ** represents <i>p</i><0.05). (B) MCF7 cells were transfected and treated in the same manner as in (A). (C) HeLa cells transfected with ILK-directed siRNA or non-target siRNA were allowed to adhere to plates coated with Pluronic, collagen type I or invasin. Cells were lysed and analysed as explained above. A representative western blot is shown and the graph to the right shows quantification of AKT pSer473 levels on Pluronic and invasin (mean ± s.e.m.; <i>n</i> = 3). (D) HeLa cells were transfected simultaneously with PAK1- and PAK2-directed siRNAs or non-target siRNA. Adhesion assays were performed on plates coated with Pluronic, invasin or collagen type I. Cell lysates were subjected to SDS-PAGE (10% gel) followed by western blotting using the different antibodies as indicated. A representative western blot is shown and the graph below presents quantification of AKT pSer473 levels on Pluronic and invasin (mean ± s.e.m.; <i>n</i> = 3). (E) Adhesion assay with MCF7 cells transfected with PAK1- and PAK2-directed siRNAs or non-target siRNA, performed as described in (A).</p

    PAK is necessary for PDGF but not for EGF-mediated AKT Ser473 phosphorylation whereas RICTOR knockdown inhibits both pathways.

    No full text
    <p>(A) PAK1 and PAK2 expression was suppressed in MCF7 cells using siRNAs and the cells were stimulated with 20 ng/ml PDGF-BB for 10 min. A representative western blot is shown and AKT pSer473 is quantified below (mean ± s.e.m.; <i>n</i> = 2, ** represents <i>p</i><0.05). (B) MCF7 and HeLa cells were transfected as above and stimulated with 20 ng/ml EGF. A representative western blot is shown and below quantification of AKT pSer473 in MCF7 and HeLa cells (mean ± s.e.m.; <i>n</i> = 3) is presented. (C) HeLa cells were treated with PAK inhibitor IPA3 (30 µM) or DMSO as vehicle control and then stimulated with 20 ng/ml PDGF-BB or EGF. A representative western blot is shown. The graph provides quantification of AKT pSer473 levels, after EGF-stimulation of cells treated with IPA3 (30 µM) normalised to the pSer473 level of this protein in EGF-stimulated cells without the inhibitor (mean ± s.e.m.; <i>n</i> = 3). (D) RICTOR expression was suppressed in HeLa cells using siRNA and the cells were stimulated with EGF (20 ng/ml). Cell lysates were subjected to SDS-PAGE followed by western blotting using antibodies as indicated. A representative blot is shown and quantification of AKT pS473 levels is found below (mean ± s.e.m.; <i>n</i> = 3, ** represents <i>p</i><0.05). (E) HeLa cells transfected with RICTOR-directed siRNA were stimulated with 20 ng/ml PDGF-BB and analysed as in (D). A representative western blot is shown and the graph below shows quantification of AKT pSer473 (mean ± s.e.m.; <i>n</i> = 2, * represents <i>p</i><0.005). (F) HeLa cells, transfected with non-target or ILK-directed siRNA, were stimulated with 20 ng/ml EGF and the samples analysed as explained above. The graph shows quantification of AKT pSer473 (mean ± s.e.m.; <i>n</i> = 2).</p

    Model of estradiol action on voltage-gated K<sup><b>+</b></sup> currents.

    No full text
    <p>A, state diagram showing the model with estradiol (E) binding to open channels (O) to form the blocked state with estradiol bound (BE). Dashed box includes the voltage-dependent transitions between closed states (C1 and C2) and the open state. B, experimentally obtained raw data currents from one neuron in control conditions (<i>black curves</i>) and in the presence of 10 µM 17-β-estradiol (<i>grey curves</i>), for comparison with C. The currents were activated by voltage steps (600 ms) to indicated potentials from a holding potential of −74 mV. C, computed K<sup>+</sup> currents for control conditions (corresponding to states enclosed by dashed box in A; <i>black curves</i>), and for the presence of 10 µM 17-β-estradiol according to the model (<i>grey curves</i>). Voltage steps as in B. D, computed currents at different concentrations of 17-β-estradiol, as indicated. Voltage step to +26 mV from −74 mV. E, concentration-response curve for computed currents 600 ms after a voltage step from −74 mV to +26 mV. EC<sub>50</sub> value and Hill coefficient (<i>n</i>) are given in the figure. F, voltage dependence of the block induced by 10 µM 17-β-estradiol (600 ms after a voltage step from −74 mV to indicated potentials) according to the model. The current in 17-β-estradiol is plotted relative to control. The line is a fitted exponential curve, with an <i>e</i>-fold change in relative current per 15 mV.</p

    17-β-estradiol rapidly reduces K<sup><b>+</b></sup> currents in MPN neurons.

    No full text
    <p>A, K<sup>+</sup> currents evoked by a voltage step from −74 mV to +36 mV, in control solution, after the addition of 10 µM 17-β-estradiol and after wash-out of 17-β-estradiol, as indicated. B, time course of estradiol-induced depression, from the neuron in A. Mean current 190–200 ms after voltage steps to +26 mV from −74 mV. (No leak current subtraction.) 17-β-estradiol was applied as indicated. Superimposed lines show fitted exponentials. C, concentration-response relation for 17-β-estradiol-induced depression of K<sup>+</sup> currents. Mean currents 590–600 ms after a voltage step to +26 mV from −74 mV. Smooth line is described by Equation 1 with EC<sub>50</sub> = 9.7 µM, n = 1.2, <i>Inh</i><sub>max</sub> = 58%. Data from 7 neurons. D, I–V relations for mean current 590–600 ms after a voltage step from −74 mV to the potentials indicated, for one MPN neuron. Current in solutions as indicated. E, relation between the effect of 10 µM 17-β-estradiol (ratio of current in estradiol to that in control solution; mean current 590–600 ms after voltage step from −74 mV) and membrane voltage for 12–13 neurons. Mean±S.E.M. The superimposed line is an exponential function, with <i>e</i>-fold change per 14 mV, fitted to the data. F–G, Ca<sup>2+</sup> independence of estradiol-sensitive current. F, currents evoked by a voltage step from −74 mV to +6 mV, with extracellular solution modified as indicated. Note that estradiol (10 µM) induced a similar depression in the presence and absence of Ca<sup>2+</sup>. G, current depressed (mean current 590–600 ms after a voltage step to +6 mV) by 10 µM 17-β-estradiol added to standard extracellular solution (left bar) and to a solution with Co<sup>2+</sup> substituted for Ca<sup>2+</sup> (right bar). The same 9 neurons were used for both conditions. The difference was not significant.</p

    Effects of altered [K<sup><b>+</b></sup>]<sub>o</sub> on block by 17-β-estradiol.

    No full text
    <p>A, currents recorded at 600-ms voltage steps to −24 mV from −74 mV. At −24 mV currents in standard [K<sup>+</sup>]<sub>o</sub> of 5 mM are outward (<i>top traces, control black</i>) with some reduction caused by 10 µM 17-β-estradiol (<i>grey</i>). With a [K<sup>+</sup>]<sub>o</sub> of 140 mM, currents (<i>lower traces, control black</i>) are inward and 10 µM 17-β-estradiol (<i>grey</i>) also reduces the current. B, relative current (mean±S.E.M. from 6 cells) in 10 µM 17-β-estradiol, measured 590–600 ms after a voltage step as in A. C, currents as in A but with voltage step to +16 mV. D, relative current (mean±S.E.M. from 5 cells) in 10 µM 17-β-estradiol, measured 590–600 ms after a voltage step as in C. E, currents recorded as in C, but with comparison between standard [K<sup>+</sup>]<sub>o</sub> of 5 mM and [K<sup>+</sup>]<sub>o</sub> = 0 mM. F, relative current (mean±S.E.M. from 7 cells) in 10 µM 17-β-estradiol, measured 590–600 ms after a voltage step as in E. Note the slight, but significant difference in blocking effect recorded from the same cells when [K<sup>+</sup>]<sub>o</sub> = 5 mM and when [K<sup>+</sup>]<sub>o</sub> = 0 mM.</p
    corecore