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Results: 7

1.
FIGURE 7.

FIGURE 7. From: Cyclin E Is Stabilized in Response to Replication Fork Barriers Leading to Prolonged S Phase Arrest.

Model for involvement of cyclin E in the replication checkpoint in response to replication fork barriers. In response to replication stress, ATR mediates stabilization of cyclin E resulting in inhibition of cyclin A-Cdk2 and retention of cyclin E and Cdc6 at stalled replication forks. These events contribute to the activation of the replication checkpoint.

Xiaoyan Lu, et al. J Biol Chem. 2009 December 18;284(51):35325-35337.
2.
FIGURE 2.

FIGURE 2. From: Cyclin E Is Stabilized in Response to Replication Fork Barriers Leading to Prolonged S Phase Arrest.

MMC inhibits ubiquitylation of cyclin E. A, Northern blot showing that MMC does not affect transcript levels of cyclin E. HeLa cells were synchronized by double-thymidine block and analyzed at the indicated times after release into normal medium with or without MMC. B, immunoblot analysis showing that MMC prevents ubiquitylation of cyclin E. HeLa cells transiently expressing HA-ubiquitin (HA-Ub) were synchronized and released into medium with or without MMC. Lysates were subjected to IP with anti-HA and immunoblotted for cyclin E (left panel). Right panel shows a loading control. C, immunoblot analysis showing stabilization of cyclin E from the experiment in B.

Xiaoyan Lu, et al. J Biol Chem. 2009 December 18;284(51):35325-35337.
3.
FIGURE 6.

FIGURE 6. From: Cyclin E Is Stabilized in Response to Replication Fork Barriers Leading to Prolonged S Phase Arrest.

ATR, but not Chk1 or Nbs1, is required for the stabilization of cyclin E in the presence of MMC. A, HeLa cells transfected with ATR, Chk1, Nbs1, or control siRNAs were synchronized and released into regular medium with MMC or without drug. Cyclin E levels were examined at the indicated times after release by immunoblot analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a loading control. B, ATR prevents the ubiquitylation of cyclin E in response to MMC. Synchronized HeLa cells depleted of ATR by siRNA (left panel) were transfected with HA-ubiquitin and subsequently subjected to IP analysis by immunoblotting (center panel). Right panel shows the loading control.

Xiaoyan Lu, et al. J Biol Chem. 2009 December 18;284(51):35325-35337.
4.
FIGURE 5.

FIGURE 5. From: Cyclin E Is Stabilized in Response to Replication Fork Barriers Leading to Prolonged S Phase Arrest.

Stabilized cyclin E and Cdc6 are maintained on chromatin in the presence of MMC. A, FACS analysis showing BrdUrd incorporation in HeLa cells released into regular medium with or without MMC (left panel). At each indicated time point cells were pulsed for 30 min with BrdUrd and then harvested for analysis. The percentage of BrdUrd-positive cells is quantitated as shown in the graph (right panel). NT, no treatment. B, cells treated as described in A were fractionated into soluble (S) and chromatin (P) fractions and subsequently analyzed by immunoblotting. C, immunoblot analysis of total lysates of samples shown in B. D, depletion of cyclin E prevents stabilization of Cdc6 in response to MMC. Cells depleted of cyclin E by siRNA were synchronized and released into MMC for the indicated times, and cellular lysates were examined by immunoblotting.

Xiaoyan Lu, et al. J Biol Chem. 2009 December 18;284(51):35325-35337.
5.
FIGURE 1.

FIGURE 1. From: Cyclin E Is Stabilized in Response to Replication Fork Barriers Leading to Prolonged S Phase Arrest.

Cyclin E is stabilized in response to replication fork barriers. A, cell cycle analysis of HeLa cells synchronized by a double-thymidine block and released into MMC. FACS analysis (left panel) and quantitation of PI staining (right panel) are shown. The highest value for the largest peak (mode) is represented. B, immunoblots showing cyclin E levels in cells presented in A. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a loading control. Far right panel shows quantitation of cyclin E bands. C and D, similar experiment as shown in A except that cells were treated with UV or IR and subsequently released into normal medium. Upper panels show FACS analysis, and lower panels show immunoblots. E, unsynchronized MRC5 primary human fibroblast cells were exposed to MMC and analyzed by FACS (left panel) and immunoblotting (right panel).

Xiaoyan Lu, et al. J Biol Chem. 2009 December 18;284(51):35325-35337.
6.
FIGURE 4.

FIGURE 4. From: Cyclin E Is Stabilized in Response to Replication Fork Barriers Leading to Prolonged S Phase Arrest.

Cdk2 kinase activity is reduced in the presence of MMC. A, upper panels, analyses showing the results of IP-kinase assays of Cdk2 using histone H1 as the substrate. HeLa cells were synchronized and released into regular medium with or without MMC for the indicated times. The top rows show autoradiographs of phosphorylated histone H1. The next two rows shown Coomassie Blue staining of histone H1 and the IgG used for the IP. The bottom two rows show immunoblots of cyclin E and the loading control GAPDH. Lower panels, experiment as described above except that the IP was performed with an antibody to cyclin A. B, immunoblot analysis showing the co-IP of Cdk2 and cyclin E with or without MMC treatment. C, sucrose gradient sedimentation profiles of cyclin A and Cdk2 8 h after release from synchronization with or without MMC. D, immunoblot analysis showing co-IP between cyclin A and Cdk2 8 h after release from synchronization. NT8, nontreated cells at 8 h after release; MMC8, cells released into MMC for 8 h. Control indicates an IP with a nonspecific IgG. E, immunoblot analysis showing levels of p21 and p27 after release into regular medium with or without MMC. F, sucrose gradient sedimentation profiles of p21, cyclin E, and cyclin A at 8 h after release into MMC.

Xiaoyan Lu, et al. J Biol Chem. 2009 December 18;284(51):35325-35337.
7.

FIGURE 3. From: Cyclin E Is Stabilized in Response to Replication Fork Barriers Leading to Prolonged S Phase Arrest.

Gradient sedimentation profiles of cyclin E and its binding partners in response to MMC. A, HeLa cells were synchronized and released into regular medium with or without MMC. Lysates were subsequently examined by sucrose gradient sedimentation (left panels). Quantitation of bands is shown in the right panels. L, loading of unfractionated samples. For reference, cell cycle distributions of these cells by FACS analysis are shown in the top panel. B, same as in A except cells were released into MMC. C, immunoblot analysis showing that MMC reduces the interaction between cyclin E and both GSK3β and Pin1 as determined in co-IP experiments. NT2, cells without drug released for 2 h; MMC8, cells treated with MMC (250 ng/ml) and released for 8 h. D, immunoblot showing that the levels of cyclin E, but not Pin1 or GSK3β, are altered in the presence of MMC. E, depletion of GSK3β results in stabilization of cyclin E during S phase. Upper panel shows FACS analysis of HeLa cell treated as indicated. Lower panel shows an immunoblot analysis.

Xiaoyan Lu, et al. J Biol Chem. 2009 December 18;284(51):35325-35337.

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