SHP2 is required for efficient repair of DNA damage.

<p>Mock- or SHP2-depleted HeLa cells were either untreated (t = 0 h) or irradiated with 10 Gy of IR. At different time points, the cells were fixed and stained for γ-H2AX. The number of cells with more than five γ-H2AX foci was quantified (<i>n</i> = 50; Mean±SD of three independent experiments). Representative images of the γ-H2AX staining are shown on the top.</p

CHK1 activation is compromised in the absence of SHP2.

<p>(<b>A</b>) HU-induced CHK1 activation is dependent on SHP2. HeLa cells were transfected with control, siSHP2, or siCHK1. The cells were treated with HU and harvested at different time points. Lysates were prepared and analyzed with immunoblotting. (<b>B</b>) CIS-induced CHK1 activation is dependent on SHP2. Control- or SHP2 siRNA-transfected HeLa cells were treated with CIS and harvested at different time points. Lysates were prepared and analyzed with immunoblotting.</p

Depletion of SHP2 increases the sensitivity to HU.

<p>(<b>A</b>) Transfection of SHP2 siRNA increases HU-mediated cell death. HeLa cells were transfected with control, siSHP2, or siCHK1. The cells were treated with HU and the levels of cell death were quantified under fluorescence microscopy (<i>n</i> = 200). Mean±SD of three independent experiments is shown. Representative images are shown in the lower panels. (<b>B</b>) Validation of SHP2 knockdown. Lysates were prepared from cells treated as described in panel A. The siRNA-mediated knockdown of SHP2 and CHK1 was confirmed by immunoblotting. Note that the signals of CHK1 appear to be weaker after HU treatment because of the multiple phosphorylation-dependent gel mobility shifts. (<b>C</b>) Downregulation of SHP2 in cells stably expressing SHP2 shRNA. HeLa cells that stably expressed shSHP2 were generated as described in Materials and Methods. Two shSHP2-expressing clones and the parental HeLa cells were treated with HU and harvested at different time points. Lysates were prepared and the expression of SHP2 was analyzed with immunoblotting. (<b>D</b>) Cells stably expressing shSHP2 are more susceptible to HU-induced cell death. Cells were treated as in panel C. At the indicated time points, the cells were harvested and processed for flow cytometry analysis. The levels of sub-G₁ population were quantified.</p

SHP2 is activated by multiple agents that induce DNA replicative stress.

<p>(<b>A</b>) SHP2^Tyr542 is phosphorylated after replication stress. HeLa cells were incubated with HU for 64 h. The cells were harvested and immediately boiled in SDS sample buffer to preserve SHP2 phosphorylation. Phosphorylated SHP2^Tyr542 and total SHP2 were detected with immunoblotting. Uniform loading of lysates was assessed by actin analysis. (<b>B</b>) SHP2 is activated by a variety of stresses. HeLa cells were treated thymidine (THY), aphidicolin (APH), HU, CIS (2 µg/ml), camptothecin (CAM), Adriamycin (ADR), or etoposide (ETP). The cells were harvested after 48 h. Phosphorylated SHP2^Tyr542 and total SHP2 were detected with immunoblotting. Uniform loading of lysates was assessed by actin analysis.</p

SHP2 is involved in preventing mitotic entry after DNA damage.

<p>(<b>A</b>) SHP2 is involved in maintaining the G₂ DNA damage checkpoint. HeLa cells (expressing histone H2B–GFP) transfected with either control or SHP2 siRNA were irradiated with 10 Gy of IR. The time of mitotic entry of individual cell was tracked with time-lapse microscopy (<i>n</i> = 43–49). The data of the individual cells can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049943#pone.0049943.s007" target="_blank">Fig. S7A</a>. (<b>B</b>) SHP2 is involved in maintaining the G₂ DNA damage checkpoint. Cells were treated as in panel B and harvested at different time points. Lysates were prepared and analyzed with immunoblotting to confirm the knockdown of SHP2. The phosphorylation of histone H3^Ser10 indicated that cells transfected with siSHP2 were able to enter mitosis after IR treatment. (<b>C</b>) SHP2 is phosphorylated after DNA damage. HeLa cells were irradiated with 10 Gy of IR and harvested at the indicated time points. The cell pellets were directly boiled in sample buffer and analyzed with immunoblotting. (<b>D</b>) IR-induced CHK1 activation is impaired in SHP2-depleted cells. Control, siSHP2(a)-, or siSHP2(b)-transfected cells were treated with 10 Gy of IR and harvested at the indicated time points. Lysates were prepared and analyzed with immunoblotting.</p

Clustering analysis of the 11 miRNAs was performed using DataAssist 3.0v based on ΔCt-values of the TLDA results.

<p>Upregulated miRNAs are designated by various shades of red and down-regulated miRNAs by various shades of green. Clinical phenotypes are labelled in different colours: active MTB infection (red), latent infection (blue), and healthy controls (green).</p

MicroRNAs differentially expressed in THP-1 macrophages infected with Beijing/W and non-Beijing/W clinical TB strains.

<p>^aFold difference in miRNA expression in THP-1 cells infected with Beijing/W clinical strains vs non-Beijing/W strains.</p><p>^bP-value was calculated by Mann-Whitney test.</p><p>MicroRNAs differentially expressed in THP-1 macrophages infected with Beijing/W and non-Beijing/W clinical TB strains.</p

Previously reported microRNAs with differential expression related to current study of MTB infections and their validated transcript targets.

<p>Previously reported microRNAs with differential expression related to current study of MTB infections and their validated transcript targets.</p

Biological pathways potentially affected by the differentially expressed microRNAs of significance from macrophages of active MTB disease, LTBI and healthy controls.

<p>Biological pathways potentially affected by the differentially expressed microRNAs of significance from macrophages of active MTB disease, LTBI and healthy controls.</p

miRNAs expression level in the THP-1 macrophages infected with Beijing/W and non-Beijing/W clinical TB strains.

<p>The relative quantity (RQ, 2-ΔΔCt) was used to normalize the relative gene expression data. Statistical analysis between two groups was performed using Mann-Whitney test. Individual values were denoted by black dots/squares from each group of Beijing/W versus non-Beijing strains. The mean RQ and S.D. of each group were represented by the ------- bar and short bars --- in each figure, respectively.</p