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These studies were undertaken to test the possibility that phosphorylation by GSK3 …


Biology Articles » Cell biology » Glycogen synthase kinase 3 has a limited role in cell cycle regulation of cyclin D1 levels » Figures

Figures
- Glycogen synthase kinase 3 has a limited role in cell cycle regulation of cyclin D1 levels

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Figure 1 Cyclin D1 expression following inhibitor treatment: (A) MRC5 cells were left untreated (left) or were treated with 20 μM LY294002 (middle) to inhibit PI3K or 50 nM rapamycin (right) to inhibit mTOR. After 2 hrs cyclin D1, BrdU added as a final pulse, and DNA were stained and subjected to image analysis. Cyclin D1 levels for individual cells are plotted vs. DNA levels, with BrdU positive, S phase cells shown as small, solid circles; with cell cycle phases noted. (B) Serum was removed from NIH3T3 cultures for 4 hrs, after which cells were fixed and stained for total cyclin D1 and with an antibody able to recognize phospho-Thr-286 cyclin D1. The average cyclin D1 (left) or phospho-Thr-286 cyclin D1 (middle) for each cell cycle phase was determined and plotted (darker bars), together with the similar value determined from a parallel culture maintained in serum (lighter bars). Finally (right) the ratio of phospho-cyclin D1 to total cyclin D1 is presented for both the normal and serum deprived culture.

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Figure 2 Dominant inhibitory AKT and cyclin D1 expression: (A) A plasmid expressing dominant inhibitory AKT was microinjected into NIH3T3 cells at the left of the area shown. The cells were incubated 8 hrs, fixed and stained with a fluorescent antibody stain against cyclin D1 (top), AKT (middle), or with DAPI to stain DNA (bottom). The same area of cells is shown in each frame. (B) NIH3T3 cells were injected with the inhibitory AKT plasmid 24 hrs prior to fixation and staining as above. Injected and uninjected cells were identified by staining for AKT, and cells were divided into cell cycle phase according to BrdU labeling and DNA content. The average cyclin D1 levels are shown for each group of cells.

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Figure 3 PI3K activity through the cell cycle. NIH3T3 cells were deprived of serum for approximately 30 hrs and injected with the PH-AKT-GFP plasmid. 15 hrs later cells were photographed under confocal microscopy either 20 min following serum stimulation (A), or without stimulation (B). Similarly, (C) cycling NIH3T3 cells were injected with the plasmid and photographed 15 hrs later. To confirm that the cycling cells retained the ability to stimulate PI3K activity, they were injected with the plasmid and deprived of serum for 0 hr (D), 5 hrs (E), 9 hrs (F) or 12 hrs (G) prior to addition of serum 20 min before confocal microscopy. Note the concentrations of fluorescence in cytoplasmic projections (E, F) or uniformly over the plasma membrane (G) following serum stimulation. The appearance of stimulated cells is clearly illustrated in a complete series of confocal sections at varying magnifications [presented in Additional files, movies 1, 2, 3].

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Figure 4 AKT activity in S and G2 phases: (A) NIH3T3 cells were synchronized with thymidine treatment in S phase and released by thymidine removal for the indicated times. In addition, quiescent cells were left unstimulated (G0), or stimulated with serum for the indicated times. In each case, lysates were collected and probed with an antibody specific for the activating phosphorylation on AKT. This was compared to western analysis of total AKT protein. Cells enter G2 phase 4–5 hrs following thymidine removal. (C) The levels were quantitated, and the ratio of phospho-specific AKT divided by total AKT levels is presented. (B) For comparison, the levels of cyclin D1 were analyzed by western analysis in the same lysates, (D) and quantitated.

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Figure 5 GSK3 activity does not vary between S and G2 phases: NIH3T3 cells were synchronized with thymidine treatment and released by thymidine removal for the indicated times. Cells remain in S phase for the first four hrs after thymidine removal and then enter G2 phase thereafter. (A) Cell lysates were collected at the indicated times following thymidine removal, and subjected to Western analysis for GSK3β phosphorylated on position 9, together with total GSK3 levels (upper panel). The results were quantitated, and the ratio of phosphorylate to non-phosphorylate GSK3 is presented (lower panel). (B) GSK3 protein was immunoprecipitated from cell lysates collected at the indicated times following thymidine release and the GSK3 protein assayed for kinase activity against a synthetic substrate. This figure represents the combined results of 10 different analyses, normalized to the level of GSK3 activity in thymidine-blocked cells. For comparison, S phase lysates were treated with 50 mM LiCl.

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Figure 6 Inhibition of GSK3 does not alter cyclin D1 levels: (A) MRC5 cells were treated for 3 hrs with MG132 alone, or in combination with 25 mM LiCl. The cells were then fixed and the level of cyclin D1 (left), or the level of phospho-Thr-286 cyclin D1 (right), for each cell was determined by image analysis and plotted against its level of DNA (with BrdU positive cells indicated as small, closed circles). (B) In an analogous experiment, MRC5 cells were treated with the indicated concentration of LiCl together with MG132. After 3 hrs the average level of phospho-Thr-286 cyclin D1 in each cell cycle phase was determined for each LiCl concentration indicated (0, 12.5, of 25 mM). (C) NIH3T3 cells were treated with MG132 together with 25 mM valproate (V), with 25 μM GSK3 inhibitor II (II), or with no treatment as a control (C). The average levels of phospho-Thr-286 cyclin D1 in each cell cycle phase of each treatment is shown.

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Figure 7 LiCl inhibits phosphorylation of β-catenin but not cyclin D1: NIH3T3 cells were cultured in medium containing 25 mM LiCl (columns 3, 4), 50 mM LiCl (column 5), or control medium with NaCl (columns 1, 2). In some cultures MG132 was added to block degradation of phosphorylated proteins (columns 2, 3, 5; see legend at the top). After 4 hrs lysates were prepared and probed for β-catenin, β-catenin phosphorylated on Ser 33/37/Thr 41, cyclin D1, cyclin D1 phosphorylated on Thr-286, and for actin as a loading control. (A) Photographs of the western results are presented. Each band was then quantitated, and (B) the ratio of phosphorylated cyclin D1 divided by the level of total cyclin D1 protein was determined and plotted immediately below the corresponding western bands. In addition, (C) the ratio of phosphorylated β-catenin divided by the level of total β-catenin protein is presented. Quantitative results correspond to the bands displayed in this figure immediately above them (with the same lane numbers).

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Figure 8 siRNA against GSK3 does not influence cyclin D1 phosphorylation: (A) A vector expressing activated GSK3β protein was injected into an NIH3T3 plate within a circular area marked on the back of the coverslip and indicated in the photographs above. (B) A similar injection of GSK3β was preceded 15 hrs earlier with an injection of an siRNA mixture against GSK3 α and GSK3 β. 5 hrs after these GSK3 plasmid injections cells were fixed and the GSK3 stained with a fluorescent antibody stain. The fluorescence photographs of each injection are presented. (C) To determine the influence of reduced GSK3 expression on the phosphorylation of cyclin D1, this siRNA was microinjected 15 hrs prior to a pulse with BrdU, fixation and staining for phospho-Thr-286 cyclin D1, BrdU and DNA. The average phospho-Thr-286 cyclin D1 levels in each cell cycle phase of injected, or neighboring uninjected cells is presented. MG132 was added 2 hrs prior to fixation to preserve phosphorylated cyclin D1 as above.

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Figure 9 Activated GSK3 does not alter cyclin D1 levels: (A) A plasmid expressing an activated mutant of GSK3β was microinjected into the NIH3T3 cells at the right side of the area of cells photographed. 8 hrs following injection the cells were fixed and stained for GSK3, cyclin D1 and DNA. Separate fluorescence photographs of the same area of cells for of each fluorochrome are presented. (B) An injection of the GSK3 plasmid into NIH3T3 cells as above was performed and the expression of GSK3 determined by image analysis and plotted vs. the level of cyclin D1 in each cell. Injected cells are shown as small, solid circles. (C) An injection as described above was performed, but the cells were treated with MG132, and stained for phospho-Thr-286 cyclin D1 and BrdU. The average levels of phospho-cyclin D1 are plotted for each cell cycle phase for injected and neighboring uninjected cells.

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Figure 10 GSK3 regulation of cyclin D1 expression in the absence of serum: NIH3T3 cells were cultured in the indicated medium, following an initial pulse with BrdU. After 11 hrs the cells were fixed, stained and analyzed. (A) The cyclin D1 level of each cells is plotted vs. its DNA content, but only cells labeled with BrdU, and therefore in S phase at the beginning of the treatment period are displayed. (B) Three identical experiments were performed and the average level of cyclin D1 in G2 phase cells following all treatments is presented. For this analysis the value for serum-deprived cells was set at 1.0, and the relative expression levels following other treatments is displayed.

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