Diagram summarising the key library construction steps.

<p>1. Gateway cloning was used to transfer ORFs into the lentiviral expression plasmid plv411G downstream of the EF1α promoter and upstream of the IRES driven GFP; 2. Virus production was performed by transfecting expression clones together with viral packaging plasmids into HEK293T cell line; 3. Viral supernatant was collected into a fresh set of plates and stored; 4. Transfected cells remaining in the plates were fixed and scanned to determine GFP fluorescence in each well. Frequency distribution histogram illustrates number of wells (y-axis) that had similar GFP fluorescence intensity (x-axis). Values between plates were normalized by zeroing on the mean of 4 mock transfected wells on each plate. Each plate also contained 4 empty expression vector wells, shown in green, which were used as positive controls; 5. Thirty eight ORF-expressing wells were randomly selected from each of the tail ends of the frequency distribution categories, to evaluate the performance of the viral supernatants on a range of target cell lines.</p

Transduction rates observed with low and high titer virus-producing expression clones.

<p>A,C – low titer clones; B,D –high titer clones. The bars in the inset in graph A represent mean and standard deviation (error bars) of four wells transduced with empty vector virus. Color of bars corresponds to the target cell line as indicated in the inset graph. Actively dividing human epithelial tumor (MCF7, PMC42-ET, MDA-MB-468, WMM1175), and non-tumor (MCF10A, HaCaT) cell lines were compared to non-dividing cells (WMM1175-p16, arrested by induced overexpression of p16, and primary mouse bone marrow macrophages (mBMM)). The proportion of GFP positive cells (y-axis) was determined after high-content imaging of plates containing fixed transduced cells. Each bar in A and B represents data for a well transduced with a single gene-expressing vector as indicated on x-axis. Bars representing mean values for vector wells are included in all three graphs to allow for scale comparison. Each bar in C and D represents a mean (error bar  = SD) transduction rate for all human cell lines for a given gene.</p

Comparison of target cell transduction rate and GFP fluorescence of corresponding virus-producing wells.

<p>A,C – target cell transduction rates; B,D – GFP fluorescence of virus producing wells. Bars in A and B represent mean of 4 wells derived from independent single colony isolates of the gene-expressing plasmids (x-axis). Genes underlined in A, produced high viral titer in bulk well experiments (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051733#pone-0051733-g001" target="_blank">Figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051733#pone-0051733-g002" target="_blank">2</a>.), the rest were low titer producers. Error bars = SD. Data for graphs A and C were obtained by high-content imaging of transduced cells, while data for graphs B and D were obtained by scanning the transfected cells using a fluorescence plate reader. C and D: Mean and SD for high and low titer well data represented in A and B. In all cell lines mean for combined low titer genes was significantly different from the mean for high titer wells (Aspin-Welch test, P value: HEK293T = 4.70 E−11; HACAT = 5.68 E−23; MDA-MB468 = 2.32 E−36; WMM1175 = 7.29 E−12; MCF10A  = 2.98 E−21).</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

Library clone insert size and GFP fluorescence in virus-making cells.

<p>A: scatter-plot of cloned insert size and total well GFP fluorescence in transfected HEK293T cells. B: frequency distribution histogram of clones in different insert size category.</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

Effect of low virus titer-producing genes on viability of the transfected cells.

<p>HEK293T cells were transfected with gene (x-axis) expressing plasmids either alone or with viral packaging vector day after seeding. 21 h after transfection, medium was replaced with PBS containing Hoechst33342 and propidium iodide (PI), which stain DNA and dying cells respectively. Cells were scanned in blue (Hoechst, A) red (PI, B) and green (GFP, C) channels and number of objects determined in each channel. A and B represent independent object counts, data in C are expressed as proportion of GFP positive cells in the Hoechst stained population. A - Significantly reduced number of surviving cells in the well compared to untreated wells (CELLS): lipofectamine alone (MOCK) (P = 0.034); MOCK with packaging plasmids (P = 7.5E−10); all others (P≤1.1E−05) B - Significantly increased PI positive cells compared to vector: expression plasmid only BCL2L1 (P = 6.88E−05), C3ORF1(P = 0.03), MTCH1(P = 4.66E−05), PNMAL2 (P = 0.01) P values determined using Aspin-Welch test; Bars = mean of 4 wells, error bars = SD, CELLS-untreated well, MOCK-wells where expression plasmid has been omitted. AWAT2, and vector were high virus titer producing in previous experiments.</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