Excess Usp44 leads to increased cyclin B in early mitosis.

<p>(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.</p

Usp44 expression leads to reduced mitotic slippage.

<p>(a) The duration of mitotic arrest in nocodazole was determined for MEFs transduced with either empty vector or Usp44-HA (n = 40–60 cells each). Chromosomes were visualized by prior transduction with lentivirus expressing H2B-YFP. After the addition of nocodazole, cells entering mitosis (for up to 20 minutes) were marked and followed by time lapse imaging for over 9 hours. The duration of arrest was defined as the time from nuclear envelope breakdown (NEBD) to de-condensation of chromosomes and nuclear reformation. (b) The duration of mitosis was determined by live-cell microscopy of MEFs with the indicated constructs. All cells were transduced with H2B-YFP and the time from nuclear envelope breakdown (NEBD) to anaphase onset was determined. N = 79 cells with control, 84 with Usp44-HA, 21 Usp44^CherryLO, 20 Usp44^CherryHI. p-values were calculated using the 2-way ANOVA. (c) The growth of cells was determined for three independent MEF lines transduced with either empty vector, or Usp44-HA. Viable cells were counted daily in triplicate using trypan blue exclusion. (d) Immunoprecipitation of Cdc27 from MEFs with or without over-expressed Usp44-HA. Precipitated material was immunoblotted with the indicated antibodies. (e) Immunoprecipitation of Cdc20 from MEFs with or without over-expressed Usp44-HA. Two results out of six independent experiments are shown. Relative amounts of Mad2 and BubR1, normalized to the mount of Cdc27 retrieved, are shown. (f) Three independent MEF lines were transduced with the indicated constructs and mitotic cells isolated by shake-off in the presence of nocodazole. Extracts were probed with the indicated antibodies. (g) Cdc20 localization was assessed by confocal microscopy on MEF cultures transduced with the indicated constructs. Images are representative of the results seen in three independent MEF lines. Relative amounts of Cdc20, normalized to the amount of PH3, are shown. * Unless otherwise noted, p<0.05 for each indicated time point calculated with an unpaired t-test. Error bars represent the SEM.</p

Usp44 is degraded by the proteasome.

<p>(a, b) Immunoblots of MEFs synchronized either by double-thymidine block (a) or mitotic shake-off (b), expressing Usp44-HA. Cells were incubated with MG132 3 hours after double-thymidine release or one hour prior to mitotic shake off. Relative quantitation was performed using imageJ. (c) Immunofluorescence images of cells expressing Usp44-HA treated (where indicated) with MG132 one hour prior to fixation.</p

Expression of Usp44 leads to chromosome missegregation and aneuploidy.

<p>(a) Expression of Usp44-HA in MEFs as seen by immunoblotting (upper) or quantitative real-time (qRT)-PCR (lower). Four independent MEF lines were transduced with Usp44-HA or Usp44Cherry followed by TaqMan qRT-PCR. Expression was normalized to GAPDH, and fold-change was calculated using the ΔCt method. (b) MEFs stably transduced with either empty vector (n = 78 cells), Usp44-HA (n = 106 cells), or Usp44^Cherry (n = 53 total) were analyzed by live-cell microscopy through the indicated numbers of un-perturbed cell divisions. Chromosomes were visualized by transduction with lentivirus encoding histone H2B fused with yellow fluorescent protein (H2B-YFP). The results depict the average and standard error from three-independent MEF lines. (c) Example of a cell with a lagging chromosome (upper) and anaphase bridge (lower). (d) Karyotype analysis of low passage MEFs. Stable transduction with the indicated lentivirus was performed at passage 2. N (metaphases)  = 175 (Empty vector control) and 223 (Usp44-HA) from a total of four independent MEF lines per genotype. (e) Representative spectral karyotype (SKY) of a cell expressing Usp44-HA. Note this cell has a normal chromosome number (40) but has a trisomy of chromosome 12 and monosomy of chromosome 7. * p<0.05 calculated with the unpairted t-test. Error bars represent the SEM.</p

USP44 is over-expressed in T-cell acute lymphoblastic leukemia.

<p>(a) Relative USP44 mRNA expression in various cancer types versus matched normal tissue was collected from publicly available microarray studies (<a href="http://www.oncomine.org" target="_blank">www.oncomine.org</a>). For all studies shown, p<0.05 for alterations in USP44 mRNA. (b) USP44 mRNA was measured by TaqMan quantitative real-time PCR in a series of 24 samples of T-cell acute lymphoblastic leukemia compared with peripheral T-cells isolated from 10 healthy volunteers. The p value was calculated with an unpaired t-test comparing the mean fold-change of T-ALL to controls. (c) Human foreskin fibroblasts were transduced with the indicated constructs (two independent lines for each), cultured for 5-passages, and then analyzed by chromosome counting. The p value was calculated using the unpaired t test.</p

Detailed analysis of Nup358 knockout MEFs expressing the minimal Nup358 fragment, GFP1-1340.

<p><b>A</b>) Paired cell lines (17, 18,19) either wild type Nup358^F/F or stable knockout Nup358^{−/−[GFP1-1340]} cells that express GFP1-1340 were infected with a VSV-G pseudotyped single cycle reporter virus HIV-1_luc. Samples were analyzed for luciferase activity 96 hours later and values were normalized to protein concentration. Error bars indicate the standard deviation between duplicate luciferase activity measurements in each experiment. <b>B</b>) The indicated cells were infected with serial dilutions of HIV-1_luc reporter virus. <b>C</b>) Aggregate results of repeated experiments (n = 10) in stable Nup358^{−/−[GFP1-1340]} knockout cells, presented as a percentage of the values for parental Nup358^F/F cells. The mean of the ten separate infection experiments is shown. 17 and 18 refer to separate Nup358^{−/−[GFP1-1340]} stable knockout MEFs. The p-value obtained using a two tailed T test between 17 and 18 Nup358^F/F and Nup358^{−/−[GFP1-1340]} was 0.62 and 0.79 respectively. <b>D–F</b>) Nup358^F/F cells and Nup358^{−/−[GFP1-1340]} cells were transduced with luciferase-encoding retroviral vectors. (D) FIV; (E) EIAV; F) MLV. Samples were analyzed for luciferase activity 96 hours later and values were normalized to protein concentration. Error bars indicate the standard deviation between duplicate luciferase activity measurements in each experiment. <b>G</b>) Results of 2-LTR circle measurements (n = 5 experiments) in stable Nup358^{−/−[GFP1-1340]} knockout cells, presented as a percentage of the values obtained in parental Nup358^F/F cells are shown for an integration competent (WT) and an integration mutant (IN D64N) HIV-1_luc. Statistical analysis was performed using two tailed T-test, p-values were 0.038 and 0.02 for WT and D64N samples respectively.</p

HIV-1 TNPO3 dependence is the same in human and mouse cells.

<p><b>A</b>) Indicated cell lines were transfected with control siRNA or siRNA for TNP03. At 72 hours after transfection, cells were harvested and analyzed for TNPO3 protein levels. Tubulin loading control is shown also. <b>B</b>) MEFs treated with siRNAs (solid black bars: control siRNA; hatched gray bars: TNPO3 siRNA) were challenged with WT or N74D HIV-1 reporter virus. Intracellular luciferase activities were determined 72 hrs after infection. 17 and 18 refer to two independently derived MEF Nup358 knockout cell lines. P value, obtained using a two tailed T test, for the difference between control compared to TNPO3 siRNA is 0.005.</p

Acute and stable Nup358 depletion in human CD4+ T cells.

<p><b>A</b>) SupT1 cells were transduced with a lentiviral vector that co-expresses mCherry and a Nup358-targeted shRNA. Four days after transduction, cells were uniformly mCherry-positive and protein depletion was confirmed using mab414. Two separate cell lines were generated (second and third lanes of immunoblot). <b>B</b>) At the same time that cells were sampled for the immunoblotting shown in (A), they were challenged with HIV-1_luc and luciferase activities were analyzed at 48 hours after infection. <b>C</b>) Infection of Supt1 cells with HIV-1 and HIV-1 N74D capsid mutant reporter viruses after acute (left) and stable (right) knockdown. See panel 8D and associated text for description of the stable line. <b>D</b>) SupT1 cells 6 weeks after shRNA transduction with two different antibodies confirms persistent knockdown of Nup358. The two lanes in the top immunoblot are from the same film of the same gel. Note that the smaller nucleoporins detected by mAb414 confirm equal loading. <b>E</b>) Equal numbers of cells from parental SupT1 or stable shRNA knockdown cells were lysed and used in western blot for RanGAP1. In the absence of Nup358, sumoylated RanGAP-1 levels decrease, and there is an increase in un sumoylated parent. <b>F</b>) Control (black bars) and stable Nup358 knockdown cells (hatched bars) were challenged with HIV-1_luc (WT or N74D), FIV_luc, and MLV_luc. <b>G</b>) Integration levels in control (black bar) and stably Nup358-depleted SupT1 cells (hatched bars) were analyzed by Alu-PCR.</p

Verification of SupT1 knockdown in acutely and stably depleted cells and challenge with HIV-1, HIV-1 G89A, HIV-1 P90A, SIVmac, FIV, EIAV and MLV.

<p><b>A</b>) Extent, specificity and stability of shRNA knockdown. Cells frozen 1, 2, and 5 months after stable depletion by shRNA transduction were thawed, passaged for ten days and lysates were immunoblotted with mAb 414, which recognizes Nup358, Nup214, Nup153 and Nup62. Two different exposures of the left immunoblot are shown. A second immunoblot of the stable cell lines was also done (right blot). The mCherry marker co-encoded by the shRNA-transducing lentiviral vector was assessed by FACS to further verify stability of expression. These experiments were done simultaneously with the viral challenges shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003969#ppat-1003969-g009" target="_blank">Fig. 9B</a>. MW: molecular weight. C: control. A: acute. 1, 2, 5: cells assessed at 1, 2, and 5 months after stable cell line derivation. <b>B</b>) Control cells, acutely depleted cells and stably depleted cells that were assessed in panel A by immunoblotting (lanes C, A and 1 respectively) and FACS were challenged with HIV-1, HIV-1 G89A, HIV-1 P90A, SIVmac, FIV, EIAV and MLV vectors. G89V vector gave the same result as G89A vector (data not shown).</p

Western blot of ERK1/2, p-38 and HSP47.

<p><b>A)</b> Western blot of the expression of ERK1/2, p-38 and HSP47 proteins from hearts of Tg and Wt mice with and without ISO treatment. B) Quantification of the ratio of phospho−/total Erk1/2 from hearts of Tg and Wt mice with and without ISO treatment. C) Quantification of the ratio of phospho−/total p-38 from hearts of Tg and Wt mice with and without ISO treatment. D) Quantification of the HSP47 protein expression from hearts of Tg and Wt mice with and without ISO treatment. * <i>P<0.05</i>, Wt vs. Tg.</p