A) Transcription repression.

<div><p>The <i>Cyclin A-Luc</i> reporter plasmid contains a firefly luciferase expression cassette preceded with a fragment from the human <i>Cyclin A</i> promoter containing E2F-binding site <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000082#pone.0000082-Strobeck1" target="_blank">[24]</a>.</p> <p>The reporter was transfected into SAOS-2 cells with the indicated RB expression plasmid and the control Renilla luciferase reporter.</p> <p>The normalized firefly luciferase activity from vector co-transfected cells was set at 100%.</p> <p>B) GST pull-down assay.</p> <p>Lysates from 293T cells expressing HA-tagged E2F1 and DP1 were incubated with the indicated GST-RB and GST-E7 proteins immobilized on glutathione Sepharose.</p> <p>The HA-E2F1 in the bound fraction was resolved by SDS-PAGE and detected by immunoblotting with an anti-HA antibody (α-HA).</p> <p>C) Co-immunoprecipitation. The RB-deficient C33A cells were co-transfected with HA-E2F1, DP1 and the indicated RB expression plasmids.</p> <p>Whole cell lysates were incubated with anti-RB antibody (α-RB), and the co-immunoprecipitated E2F1 examined by immunoblotting with an anti-HA antibody (α-HA).</p> <p>D) GST-pull down assay with E2F3.</p> <p>Lysates from 293T cells expressing HA-tagged E2F3 and DP1 were incubated with the indicated GST-RB and GST-E7 proteins immobilized on glutathione Separose.</p> <p>The amount of E2F3 in the bound fraction was determined by immunoblotting with anti-HA antibody (α-HA).</p></div

A) Summary of RB mutants.

<div><p>The amino acid numbering is that of the human retinoblastoma protein.</p> <p>Two shaded boxes represent the A and B domains; I, the insert region; N, the region N-terminal to the A domain; C, the region C-terminal to the B domain.</p> <p>B) GST pull-down assay.</p> <p>The RB-deficient MDA-MB468 cells were infected with recombinant adenovirus encoding GFP, RB, RB-K, RB-N or RB-KN, whole cell lysates were incubated with purified GST-E2F1 or GST-E7 fusion protein immobilized on glutathione-Sepharose.</p> <p>The bound fractions were solubilized, fractionated by SDS-PAGE and reacted with an affinity-purified anti-RB.</p> <p>C) Reciprocal GST pull-down assay.</p> <p>Lysates from SAOS-2-tet-E2F1 cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000082#pone.0000082-Phillips1" target="_blank">[21]</a> were immunoblotted with anti-E2F1 to demonstrate the induced expression of E2F1 protein (upper panel).</p> <p>Lysates from induced and non-induced cells were incubated with GST-RB or GST-RB-K fusion proteins immobilized on glutathione-Sepharose, and the bound fraction analyzed by immunoblotting with anti-E2F1 (lower panel).</p> <p>D) Co-immunoprecipitation assay.</p> <p>SAOS-2-tet-E2F1 cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000082#pone.0000082-Phillips1" target="_blank">[21]</a> were infected with recombinant adenoviruses encoding GFP, RB, RB-K, RB-N or RB-KN and then cultured without tetracycline to induce the expression of E2F1.</p> <p>Co-immunoprecipitation of E2F1 in anti-RB immune complex was determined by immunblotting with anti-E2F1 antibody (α-E2F1). E) Luciferase assay.</p> <p>The Gal4-Luc reporter plasmid contains a firefly luciferase expression cassette with five Gal4 binding sequence motifs.</p> <p>The Gal4-E2F1 protein contains the Gal4-DNA binding domain and the transactivation domain of E2F1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000082#pone.0000082-Flemington1" target="_blank">[22]</a>.</p> <p>SAOS-2 cells were transfected with Gal4-Luc and Gal4-E2F1 plasmids in the presence or absence of RB or RB-K.</p> <p>A plasmid expressing Renilla luciferase from the thymidine kinase promoter was included in the transfections for normalization of transfection efficiency.</p> <p>The firefly and Renilla luciferase activities were measured in each sample, the ratio of which was compared among the different transfections with that from the Gal4-Luc only transfected sample as the baseline.</p> <p>The values shown are averages and standard errors from three independent experiments.</p></div

A) RB phosphorylation by Cdk2/Cyclin E in SAOS-2 cells.

<div><p>Cells were infected with recombinant adenovirus expressing RB or RB-KN, in the absence or presence of co-infection with recombinant adenovirus expressing Cdk2 and Cyclin E.</p> <p>The electrophoresis mobility of RB was examined by immunoblotting of whole cell lysates with anti-RB.</p> <p>B) Representative FACS profiles. SAOS-2 cells infected with the indicated recombinant adenoviruses were treated with 50 µM etoposide, harvested 48 hours later, fixed, stained with propidium iodide and the DNA content analyzed by FACS.</p> <p>The gate for sub-G1 DNA fraction is shown in each profile.</p> <p>C) Summary of DNA fragmentation results.</p> <p>The sub-G1 DNA content was determined by FACS analysis as illustrated in panel B.</p> <p>The values are mean and standard errors from three independent experiments.</p></div

A) BrdU incorporation.

<div><p>MDA-MB468 cells were infected with the indicated recombinant adenoviruses encoding each of the indicated proteins.</p> <p>Infected cells were allowed to incorporate BrdU over a period of 14 hours before they were fixed and stained with phycoerythrin (PE)-conjugated monoclonal antibody against BrdU.</p> <p>The fraction of BrdU-positive cells in each sample was measured by flow cytometry (FACS).</p> <p>The levels of BrdU incorporation in GFP-virus and RB-KN-virus infected cells were statistically similar by student <i>t</i>-test (*) from three independent experiments.</p> <p>Representative FACS profiles with the BrdU-positive gates shown are displayed to the right of the histogram.</p> <p>B) Flat cell formation. SAOS-2 cells transfected with plasmids expressing neomycin resistance and each of the indicated RB proteins were cultured in G418 for two weeks.</p> <p>The giant flat cells were stained with crystal violet and their numbers counted under a dissection microscope.</p> <p>The number of flat cells induced by RB in each of four independent experiments was set at 100%.</p> <p>The means and standard errors of relative flat cell induced by vector, RB-K, RB-N and RB-KN from four experiments are shown.</p> <p>Student <i>t</i>-test showed the number of flat cells found in RB-KN-transfected cultures was significantly different (* p<0.002) from that in RB-transfected cultures.</p></div

Anti-miRs Competitively Inhibit microRNAs in Argonaute Complexes

<div><p>MicroRNAs (miRNAs), small RNA molecules that post-transcriptionally regulate mRNA expression, are crucial in diverse developmental and physiological programs and their misregulation can lead to disease. Chemically modified oligonucleotides have been developed to modulate miRNA activity for therapeutic intervention in disease settings, but their mechanism of action has not been fully elucidated. Here we show that the miRNA inhibitors (anti-miRs) physically associate with Argonaute proteins in the context of the cognate target miRNA <i>in vitro</i> and <i>in vivo</i>. The association is mediated by the seed region of the miRNA and is sensitive to the placement of chemical modifications. Furthermore, the targeted miRNAs are stable and continue to be associated with Argonaute. Our results suggest that anti-miRs specifically associate with Argonaute-bound miRNAs, preventing association with target mRNAs, which leads to subsequent stabilization and thus increased expression of the targeted mRNAs.</p></div