Full-length IncA of <i>Cp. psittaci</i> and G3BP1 proteins interact <i>in vitro</i> and <i>in vivo</i>.

<p>(A) Purified GST, GST-IncA (aa 1-338) and GST-IncA (aa 121-383) or (C) GST and GST-G3BP1 (aa1-466) stained with Coomassie. (B) Results of GST pull-down experiments performed with IncA full-length and truncation clone fused to GST and [³⁵S]-methionine-labeled full-length G3BP1 or (E) with Hep-2 cell lysate instead of labeled G3BP1. (D) Results of GST pull-down experiment performed with full-length G3BP1 fused to GST and [³⁵S]-methionine-labeled IncA (aa 121-383). GST served as control. IVT: 10 µl of the <i>in vitro</i> translation product. Input: 25% (75 µg protein) of Hep-2 cell lysate used for the interaction assay. (F) Whole cell lysate of HEK293 cells transfected with a His-tagged codon-adapted IncA mammalian expression construct was subjected to co-immunoprecipitation experiments using anti-His antibody. Co-immunoprecipitated endogenous G3BP1 was detected with a mouse anti-G3BP1 monoclonal antibody. Controls were beads alone (c) or pre-immune serum (lgG). Input: 10% (100 µg) of the protein used for immunoprecipitation.</p

G3BP1 is accumulated around the chlamydial inclusion in <i>Cp. psittaci</i> infected Hep-2 cells.

<p>Hep-2 cells were infected with <i>Cp. psittaci</i> at a MOI of 3, and at 48 h postinfection the cells were fixed and stained with anti-chlamydial LPS (red), anti-G3BP1 (green) antibodies and DAPI (blue, nuclei), and viewed under a fluorescence (1–3) or a confocal laser-scanning microscope (4–6). Non-infected cells served as control (1–3). The anti-G3BP1 (1 and 4) and anti-LPS (5) or DAPI (2) signals were merged (3 and 6). A fraction of endogenous G3BP1 is concentrated around chlamydial inclusions (6). Bar = 20 µm.</p

Infection of Hep-2 cells with <i>Cp. psittaci</i> leads to a decrease in c-Myc protein concentration.

<p>Hep-2 cells were infected with <i>Cp. psittaci</i> at a MOI of 3, with heat-inactivated <i>Cp. psittaci</i> or chloramphenicol-treated after infection as controls, and at different hours postinfection (hpi) cells were lysed for RNA or protein analysis. (A, above) Semi-quantitative (sq) PCR shows the unchanged expression of <i>c-myc</i> mRNA in infected (middle column), infected and chloramphenicol-treated (right) and control (left) Hep-2 cells. <i>β-actin</i> served as an internal control gene. (A, below) Quantitative Real-Time (qRT) PCR. <i>c-myc</i> levels were standardized on <i>GAPDH</i>. Experiments were performed three times in duplicate. (B) Immunoblots of whole cell lysates of <i>Cp. psittaci</i> infected (middle column), infected and chloramphenicol-treated (right) and control (left) cells probed with anti-LPS, anti-c-Myc and anti-β-actin antibodies. Bar diagrams below the immunoblots represent a quantification of c-Myc protein related to β-actin protein concentration. Experiments were performed three times.</p

The decrease in c-Myc protein concentration can be ascribed to the interaction between IncA and G3BP1.

<p>(A) Schematic structure of the <i>Cp. psittaci</i> IncA (aa 121-383) double mutant (N159S; S237G) which is no longer able to interact with G3BP1 in the yeast two-hybrid system. Mutagenesis was performed in <i>S. cerevisiae</i> using error-prone PCR. (B) Yeast two-hybrid interaction assay of mutated IncA with G3BP1. Interactions were quantitatively assessed by β-galactosidase liquid culture activity assays. β-galactosidase values represent the mean numbers and standard deviations of three independent experiments. (1) GAL4 BD-IncA (aa 121-383)-EGFP + pGADT7 (negative control), (2) GAL4 BD-IncA (aa 121-383)-EGFP + GAL4 AD-G3BP1 (aa 221-451) (positive control), (3) GAL4 BD-IncA (aa 121-383, N159S; S237G)-EGFP + GAL4 AD-G3BP1 (aa 221-451). (C) Immunoblots of whole HEK293 cell lysates transfected with pcDNA3 (1), pcDNA3-HA-IncA (aa 121-383) (2) or pcDNA3-HA-IncA (aa 121-383, N159S; S237G) (3) and probed with anti-HA, anti-c-Myc and anti-β-actin antibodies. Bar diagram below the immunoblots represent a quantification of c-Myc protein concentration related to β-actin. Experiments were performed in triplicate. (D) Immunoblots of whole Hep-2 cells untreated (1), transfected with non-targeting siRNA (2) or transfected with ON-TARGET G3BP1 siRNA pool and probed with anti-G3BP1, anti-c-Myc and anti-β-actin antibodies. Bar diagrams show knock-down rate of G3BP1 and c-Myc standardized to β-actin and the <i>c-myc</i> mRNA concentrations estimated by qRT-PCR. Experiments were performed in triplicate.</p

Unique u80-metric mouse proteins for the individual assemblies.

<p>Almost all individual assemblies (52 of 59) have transcripts with an ungapped alignment covering a mouse protein by more than 80% that are not present in the other assemblies.</p

GO category distribution of the gene clusters from the final transcript set using all annotated GO terms up to the second level.

<p>GO category distribution of the gene clusters from the final transcript set using all annotated GO terms up to the second level.</p

Comparison of the proportions of correctly assembled transcripts and misassemblies.

<p>All transcripts with significant BLASTp hit against the mouse reference protein set were classified into “correct” (red), “short” (green) and “false” (blue) assembled transcripts.</p

Comparison of the public transcript set from Xu et(a) the reference-based re-assembly and (b) the non-reference-based re-assembly.

<p>The Venn diagram (a) gives an overview of the predicted unique gene loci of both data sets. More than 1/3 of the transcripts are present in both sets. The venn diagram (b) shows the gene clusters created with the wcd <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085568#pone.0085568-Hazelhurst1" target="_blank">[23]</a> tool.</p

Estimation of the number of cluster with paralogous genes.

<p>If transcripts from different cluster mapped on the same gene locus, the transcripts where counted as “multiple cluster”.</p

Next-generation RNA sequencing data from CHO cell lines analyzed.

<p>Next-generation RNA sequencing data from CHO cell lines analyzed.</p