Figure 3. Excess Usp44 leads to increased cyclin B in early mitosis.

(a) Cells were synchronized in G1/G0 by serum starvation and were then released. Nocodazole was added 23 hours after release. Samples were collected at the indicated times and were immunoblotted with the indicated antibodies. Results are representative of at least 3 independent experiments. (b) Immunofluorescence imaging of cells transduced with empty lentivirus or Usp44-HA using the indicated antibodies. The stage of mitosis was determined by DNA morphology. (c) Quantitation of cyclin B levels from (a) using imageJ. Ten cells from each of three independent MEF lines (total 30 cells per condition per stage) were analyzed. (d) Cyclin B1 mRNA was measured using qRT-PCR. Cells were synchronized as in (a) and harvested at the indicated times. (e) MEFs were stably transduced with the indicated construct and were then transfected with a construct encoding a fusion between cyclin B1 and Cerulean (CyclinB1^Cerulean). Cells were monitored by live-cell microscopy. Images were obtained every 4 minutes and quantified with imageJ. Values were normalized such that the level at nuclear envelope breakdown (NEBD) was set at 100%. The arrow with “A:” refers to the average time of anaphase observed in the cells in each condition. (f) MEFs were transduced with the indicated constructs and were fixed and stained to detect Usp44^Cherry, cyclin B1, and DNA. The levels of Usp44^Cherry and cyclin B1 were quantitated in G2 cells (n = 11–19 cells each) using imageJ. (g) MEFs transduced with empty vector were treated with MG132 for 1 hour prior to fixation. The amount of cyclin B1 was determined in G2 or early prophase using imageJ in comparison to untreated, or Usp44^Cherry transduced MEFs. Graph represents the average of 20 cells in each group from three independent experiments. * p<0.05 calculated with an unpaired t-test.