Specific down-regulation of <i>Mybbp1a</i> in wild type ES cells blocks proliferation and induces activation of caspase-3.

<p>(<b>A</b>) ES cells were infected with the <i>Mybbp1a</i>-specific shRNA3 or with an empty non-target (NT) lentivirus vector (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039723#s2" target="_blank">Materials and Methods</a>), and the proliferation rate was measured starting two days after infection (0 h) every 24 h, using a cell counter. (<b>B</b>). At the end of the experiment (72 h) the cells were lysed and the extract immunoblotted with Mybbp1a-specific antibodies (anti-p160C) using Tubulin as loading control. (<b>C, D</b>). The extracts were also immunoblotted against Oct4 (for specificity of the down-regulation) and cleaved-Caspase 3 for apoptosis using Vinculin as loading control.</p

Gene Onthology Analysis of the differentially expressed genes following <i>MYBBP1A</i>-down-regulation in HeLa cells.

<p>N = total number of differentially expressed genes within the GO cathegory.</p><p>%: percent of the total number of differentially expressed genes.</p><p><i>p-value</i>: calculated on the basis of the enrichment of the category (ie number of genes affected/number of genes in the category).</p

Timed pregnancy analysis of heterozygous <i>MYBBP1A</i>^+/− crosses.

<p>N = Total number.</p><p>NP = No PCR product.</p

Tumorigenesis by RasVal12 in NIH3T3 cells infected with an empty vector (C) or with a retrovirus carrying an shRNA specific for <i>MYBBP1A</i> (Sh3).

#<p>Total number of mice in the group.</p>○<p>volume expressed in cm³.</p>*<p>SEM = Standard Error of the Mean.</p>$<p>Student's t-test.</p

<i>MYBBP1A</i>-depletion slows the growth of HeLa cells.

<p>(<b>A</b>) Measure of the length of mitosis with Time-Lapse video imaging of HeLa cells transfected with High-GC or siRNA1 for 48 h after synchronization with a double thymidine block. The numbers below the histograms identify groups of cells with different mitosis length. Cells analyzed = 40 per group. (<b>B</b>) Percentage of dividing, not dividing or dead cells during the period analyzed by Time-Lapse microscopy; individual cells analyzed = 60.</p

<i>MYBBP1A</i> down-regulation induces apoptosis.

<p>(<b>A</b>) HeLa cells were transiently transfected with siRNA1, 2 or 3, or with control High-GC or Medium-GC oligonucleotides, or with Lipofectamine only, for 48 h. The figure shows the determination of early apoptotic cells by flow cytometry (i.e. Annexin V positive and 7AAD-negative) (left panel, circled gate) 48 h post transfection. The histogram on the right shows the quantification in the various samples at the different times after transfection. In untreated cells only 1% of the cells were in apoptosis. (<b>B</b>) Western-blot analysis of HeLa cells transiently transfected for 48 h with CTL (untreated cells), LIPO (treated only with lipofectamine), HIGH GC (transfected with High-GC control), siRNA1 (transfected with MYBBP1A-specific siRNA1). The immunoblot was performed against MYBBP1A, active Caspase 3 and Caspase 9; tubulin is shown as loading control.</p

Genes differentially expressed in <i>MYBBP1A</i>-down-regulated HeLa cells^*.

*<p>Data obtained with a human Affymetrix ST 1.0 chip. Analysis of gene expression profiling on HeLa cells transfected with siRNA3, harvested 48 h after transfection.</p>§<p>Genes whose expression level varied by more than 50% (p<0.0001).</p

Down-regulation of <i>Mybbp1a</i> in primary wild type MEFs and in NIH3T3 cells has opposite effects on cell proliferation.

<p>(<b>A</b>) Down regulation of <i>Mybbp1a</i> in MEFs with the specific lentiviral vector shRNA3 induces early senescence, as opposed to a non-target empty (NT) vector. (<b>B</b>) Immunoblot of the cells of panel A showing down regulation of MYBBP1A (p160), using tubulin as control. (<b>C</b>) Growth rate determination (crystal violet assay, see Methods) in Mybbp1a down-regulated NIH3T3 cells with lentiviral vectors expressing specific Sh1RNA or Sh3RNA, as opposed with control empty Non Target vector (NT). Crystal violet assays were performed over 10 days counting the cells in triplicate every 2 days. The p-value of the difference between Sh3 and NT-treated cells was ≤0.001 (t test). (<b>D</b>) Immunoblot of the cells of panel C. (<b>E</b>) Growth rate determination of <i>MYBBP1A</i> down-regulated HeLa cells by specific siRNAs (see Methods). Ctl: untreated cells. Lipo: transfection control with lipofectamine only. High GC and Medium GC: two control oligonucleotides (indicated by the siRNAs manufacturer) of high and, respectively, medium GC content. siRNA1, siRNA2 and siRNA3: specific MYBBP1A siRNAs (see Methods for sequences). (<b>F</b>) Immunoblots of the cells of panel B upon culturing for 24, 48, 72 and 96 h after transfection.</p

Connection between <i>MYBBP1A</i>, cell cycle genes and their function.

<p>The figure depicts the connection between <i>MYBBP1A</i>, the genes regulating the cell cycle that are altered by <i>MYBBP1A</i> down-regulation and their function. In particular, the expression of red genes is increased in the absence of MYBBP1A, while those in blue are decreased. Thus, MYBBP1A induces the inhibitors of Cdc2/cyclinB and down-regulates the activators. Note that although the figure focuses on the G2/M phase, some of the genes function also at the G1/S (in particular CDKN1A). MYBBP1A might act by regulating the synthesis and/or activity of one or more of its partner transcription factors (cMyb, cJun, NF-kB). In fact, the level of cJun mRNA is increased upon <i>MYBBP1A</i> down-regulation (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039723#pone-0039723-t004" target="_blank">Table 4</a>).</p

Down-regulation of <i>Mybbp1a</i> in NIH3T3 cells favors transformation by RasV12.

<p>(<b>A</b>) Soft agar colony assay with NIH 3T3 cells infected with non-target vector (NT), shRNA1 or shRNA3 lentiviruses with or without co-infection with pBABE-Ras^Val12 retrovirus. 50,000 cells were plated in triplicate in soft agar and the images recorded 9 days after plating. The left panel shows examples of the actual plates, the right panel the quantification of the experiment. (<b>B</b>) Immunoblot analysis of the levels of Ras and Mybbp1a after infection with pBABE-RavV12 and SH3 or NT (empty, non target control) lentivirus, respectively. The apparent difference in the level of Ras in this blot is due to a difference in loading (see the actin band). (<b>C</b>) Enhanced tumor growth rate upon Mybbp1a depletion. Cells were injected in the flank of nude mice (n = 6/group) and the size of the tumor evaluated at different times thereafter (ordinates, days). NIH3T3 cells were infected with non-target empty lentivirus vector (NT) alone (○), non-targeting lentivirus vector (NT) plus pBABE-RasV12 (•), <i>Mybbp1a</i>-specific shRNA3 lentivirus vector alone (□) or <i>Mybbp1a</i>-specific shRNA3 lentivirus vector plus pBABE-RasV12 (▪). At each time point, the Student's t-test gave a p-value of 0.0188 (11 days), 0.0334 (13 days), 0.035 (15 days) and 0.048 (18 days). This panel shows a single experiment, representative of a total of three, with the same result.</p