Fbxo7 cooperates with p53 mutation to promote lymphomagenesis <i>in vivo</i>.

<p>(<b>A</b>) Graph of Kaplan-Meier survival curve of mice reconstituted with p53 null FL cells infected with retroviruses expressing either MSCV control (n = 10, dashed line) or Fbxo7 (n = 10, solid line). (<b>B</b>) H&E staining and immunohistochemistry was conducted as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021165#pone.0021165-Laman1" target="_blank">[11]</a> for (<b>C</b>) CD3 and (<b>D</b>) Fbxo7 in tissue samples from mice reconstituted with Fbxo7-expressing cells. Size bar is 50 µm. (<b>E</b>) PCR amplification reactions for either the p53 null allele (top) or the GFP gene in the MSCV vector (bottom) performed on genomic DNA isolated from paraffin-embedded tissue samples from mice reconstituted with p53 null HSPCs infected with retroviruses bearing either the empty MSCV vector or the MSCV vector expressing Fbxo7. Numbered samples consist of pooled biopsies from multiple organs (liver, spleen, thymus, intestine, kidney, heart) from mice in the two different cohorts, as indicated. ‘T’ denotes samples which consisted of tumour tissue only. For positive controls for the PCR reactions, in the p53 reaction (top), ‘pos’ denotes a reaction where genomic DNA from a p53 null mouse was added, and for the GFP reaction (bottom) ‘pos’ denotes a reaction where MSCV plasmid DNA was added. ‘Neg’ denotes reactions where no template DNA was added.</p

Fbxo7 expression reduced the colony forming capacity and number of WT and p53 null cells.

<p>(<b>A</b>) Representative FACS plots of retrovirally infected FL cells showing GFP expression. (<b>B</b>) Expression of Fbxo7 in cells assayed by immunoblotting and Ponceau S staining as a loading control. (<b>C</b>) Table of quantification of the number of colonies at each passage. Number in parentheses is the percent decrease relative to the MSCV control. Error is represented as the SD. Quantification is of two independent experiments for each cell type. (<b>D</b>) Graphs of the quantification of total numbers of either WT (left) or p53 null (right) cells at each passage. Error is represented as the SD. Quantification is of two independent experiments for each cell type. * denotes statistical significance, <i>P</i> value<0.05; ** <i>P</i> value<0.01.</p

Fbxo7 expression enhances the proliferation of p53 null cells grown at a reduced SCF concentration.

<p>(<b>A</b>) FACS plot of EdU incorporation, showing forward scatter (FSC) on the x-axis and (SSC) side scatter on the y-axis for the left hand panels, with the gated population used for EdU analysis and anti-EdU conjugated to Alexafluor647 on the x-axis and propidium iodide intensity on the y-axis for the right hand panels. (<b>B</b>) Immunoblotting for the expression of various cell cycle regulators, as indicated, in WT and p53 null cells expressing Fbxo7 or the empty MSCV vector. (<b>C</b>) Immunoblotting for Cdk6 in immunoprecipitations of D-type cyclins from equal numbers of sorted WT cells expressing either Fbxo7 or the empty MSCV vector.</p

Fbxo7 acts as a proliferative factor in p53 nulls grown in reduced SCF.

<p>(<b>A</b>) Table of the quantification of colony number of WT and p53 null cells grown at different concentrations of SCF. (<b>B</b>) Graphs of the total cell number at three concentrations of SCF in WT and p53 null cells expressing Fbxo7 compared to MSCV. (<b>C</b>) Graph of ratio of the number of either WT or p53 null cells expressing Fbxo7 compared to MSCV at different concentrations of SCF. In these experiments, the error is represented as the SD, and quantification is of three independent experiments.</p