Search for massive resonances decaying in to WW,WZ or ZZ bosons in proton-proton collisions at root s=13 TeV

Peer reviewe

Search for anomalous Wtb couplings and flavour-changing neutral currents in t-channel single top quark production in pp collisions at root s=7 and 8 TeV

Peer reviewe

Measurement of prompt and nonprompt J/psi production in pp and pPb collisions at root s(NN)=5.02 TeV

Peer reviewe

Measurement and QCD analysis of double-differential inclusive jet cross sections in pp collisions at √s = 8 TeV and cross section ratios to 2.76 and 7 TeV

A measurement of the double-differential inclusive jet cross section as a function of the jet transverse momentum pT and the absolute jet rapidity |y| is presented. Data from LHC proton-proton collisions at √s= 8 TeV, corresponding to an integrated luminosity of 19.7 fb−1, have been collected with the CMS detector. Jets are reconstructed using the anti-kT clustering algorithm with a size parameter of 0.7 in a phase space region covering jet pT from 74 GeV up to 2.5 TeV and jet absolute rapidity up to |y| = 3.0. The low-pT jet range between 21 and 74 GeV is also studied up to |y| = 4.7, using a dedicated data sample corresponding to an integrated luminosity of 5.6 pb−1. The measured jet cross section is corrected for detector effects and compared with the predictions from perturbative QCD at next-to-leading order (NLO) using various sets of parton distribution functions (PDF). Cross section ratios to the corresponding measurements performed at 2.76 and 7 TeV are presented. From the measured double-differential jet cross section, the value of the strong coupling constant evaluated at the Z mass is αS(MZ) = 0.1164− 0.0043+ 0.0060, where the errors include the PDF, scale, nonperturbative effects and experimental uncertainties, using the CT10 NLO PDFs. Improved constraints on PDFs based on the inclusive jet cross section measurement are presented

Nox4 and duox1/2 mediate redox activation of mesenchymal cell migration by PDGF

Platelet derived growth factor (PDGF) orchestrates wound healing and tissue regeneration by regulating recruitment of the precursor mesenchymal stromal cells (MSC) and fibroblasts. PDGF stimulates generation of hydrogen peroxide that is required for cell migration, but the sources and intracellular targets of H2O2 remain obscure. Here we demonstrate sustained live responses of H2O2 to PDGF and identify PKB/Akt, but not Erk1/2, as the target for redox regulation in cultured 3T3 fibroblasts and MSC. Apocynin, cell-permeable catalase and LY294002 inhibited PDGF-induced migration and mitotic activity of these cells indicating involvement of PI3-kinase pathway and H2O2. Real-time PCR revealed Nox4 and Duox1/2 as the potential sources of H2O2. Silencing of Duox1/2 in fibroblasts or Nox4 in MSC reduced PDGF-stimulated intracellular H2O2, PKB/Akt phosphorylation and migration, but had no such effect on Erk1/2. In contrast to PDGF, EGF failed to increase cytoplasmic H2O2, phosphorylation of PKB/Akt and migration of fibroblasts and MSC, confirming the critical impact of redox signaling. We conclude that PDGF-induced migration of mesenchymal cells requires Nox4 and Duox1/2 enzymes, which mediate redox-sensitive activation of PI3-kinase pathway and PKB/Akt

PDGF stimulates redox-sensitive phosphorylation of PKB/Akt in mesenchymal cells, whereas EGF has no effect.

<p>(<b>A</b>)–(<b>B</b>), representative western blots showing the effects of apocynin (<b>A</b>) or PEG-catalase (<b>B</b>) on phosphorylation kinetics of PKB/Akt and Erk1/2 in PDGF-stimulated 3T3 fibroblasts, and vinculin staining in the same lysates used for the loading control. (<b>C</b>)–(<b>D</b>), the corresponding changes in phosphorylation of PKB/Akt or Erk1/2 in fibroblasts analyzed in 4 independent experiments by normalization of phosphorylation signals exemplified above to the vinculin content. Additionally, each data set was normalized to the value of 10 min stimulation in uninhibited control, which therefore has no error bar; (*) p < 0.05 as compared to uninhibited controls. (<b>E</b>)–(<b>F</b>), representative western blots showing the effects of apocynin (<b>E</b>) or PEG-catalase (<b>F</b>) on phosphorylation kinetics of PKB/Akt and Erk1/2 in PDGF-stimulated MSC, and vinculin staining in the same lysates used for the loading control. (<b>G</b>)–(<b>H</b>), the corresponding changes in phosphorylation of PKB/Akt or Erk1/2 in MSC analyzed in 4 independent experiments by normalization of phosphorylation signals exemplified above to the vinculin content. As above, each data set was normalized to the value of 10 min stimulation in uninhibited control; (*) p < 0.05 as compared to uninhibited controls. (<b>I</b>)–(<b>J</b>), PDGF, but not EGF stimulates phosphorylation of PKB/Akt in 3T3 fibroblasts (<b>I</b>) and MSC (<b>J</b>), but both PDGF and EGF similarly stimulate phosphorylation of Erk1/2. Shown are representative membranes from 2 independent experiments.</p

Inhibitory analysis of PDGF-stimulated mesenchymal cell migration.

<p>(<b>A</b>) Migration of NIH-3T3 fibroblasts. Shown are the mean cell velocities ± SE from 7 independent experiments and 225–340 cells analyzed. (*) p < 0.05 as compared to the unstimulated vehicle control, (#) p < 0.05 as compared to PDGF-stimulated control. (<b>B</b>) Migration of MSC. Shown are the mean cell velocities ± SE from 4 independent experiments and 120–200 cells analyzed. (*) p < 0.05 as compared to the unstimulated vehicle control, (#) p < 0.05 as compared to the PDGF-stimulated control.</p

PDGF, but not EGF stimulates accumulation of H₂O₂ in fibroblasts.

<p>NIH-3T3 fibroblasts were transiently transfected with the plasmid encoding HyPer-NES, the ratiometric cytoplasmic sensor for H₂O₂. (<b>A</b>) and (<b>B</b>) show typical confocal images of the control and apocynin pre-treated cells, respectively, taken at the indicated time points after stimulation with PDGF. The <i>upper</i> and <i>middle</i> image rows show the changes in fluorescence intensity excited by either 405 nm (top) or 488 nm (middle) lasers. The lower row represents ratio images that report relative changes in cytoplasmic H₂O₂. PEG-catalase (40 units/ml) was added 30 min after PDGF to decompose H₂O₂ and detect the baseline of the HyPer fluorescense ratio in cytoplasm. Scale bar, 10 μm. (<b>C</b>), the time course of H₂O₂ accumulation in the control (<i>grey</i>) and apocynin treated (<i>black</i>) cells. Shown are mean values of the HyPer fluorescence ratio ± SE obtained from 38 control and 65 apocynin-treated cells in 4 independent experiments; (*) p < 0.05 as compared to untreated controls. (<b>D</b>)-(<b>Е</b>), 3T3 fibroblasts were consequently treated with 20 ng/ml EGF and 10 ng/ml PDGF to directly compare the H₂O₂ responses. (<b>D</b>) shows confocal images taken at the indicated time points after EGF and PDGF addition depicted underneath. Scale bar, 10 μm. (<b>E</b>) shows the time course of H₂O₂ accumulation in cytoplasm; addition of growth factor is indicated by arrows. Shown are the mean values of HyPer ratio ± SE from 43 cells analyzed in 3 independent experiments.</p

Duox1/2 and Nox4 mediate the PDGF-induced responses in mesenchymal cells.

<p>Representative blots are shown on the left, statistics are shown on the right. The data are normalized to 10 min of stimulation in scrambled/NT controls for fibroblasts and 30 min of stimulation for MSC, which have therefore no error bars; (*) p < 0.05 as compared to scrambe (<i>scr</i>) or non-targeting (<i>NT</i>) controls from 2–4 independent experiments. (<b>A</b>) PDGF-induced phosphorylation of PKB/Akt and Erk1/2 in 3T3 fibroblasts stably expressing the indicated shRNAs. (<b>B</b>) PDGF-induced phosphorylation of PKB/Akt and Erk1/2 in 3T3 fibroblasts transiently transfected by indicated siRNAs. (<b>C</b>) PDGF-induced phosphorylation of PKB/Akt and Erk1/2 in MSC transiently transfected by indicated siRNAs. (<b>D</b>) Kinetics of cytoplasmic H₂O₂ accumulation in 3T3 fibroblasts pre-treated by indicated siRNAs; PDGF added at 0 min. (<b>E</b>) Effects of Duox1/2 silencing on speed of 3T3 fibroblast migration. Shown are the results of shRNA- and siRNA-mediated silencing of Duox1/2 compared, respectively, to scramble and NT controls, which moved with identical speeds.</p

Expression profile and silencing of NADPH-oxidases in mesenchymal cells.

<p>(<b>A</b>)–(<b>B</b>), RT-PCR of NADPH-oxidases in 3T3 fibroblasts and MSC, respectively. Nox5 was not assessed in 3T3 fibroblasts, because it is absent in these cells [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154157#pone.0154157.ref030" target="_blank">30</a>]. (<b>C</b>)–(<b>D</b>), 3T3 fibroblasts were stably infected by shRNAs to Nox4 or Duox1 and analyzed for corresponding mRNA (<b>C</b>) and protein expression (<b>D</b>). The graph shows mRNA expression levels normalized to those in cells expressing scrambled shRNA; (*) p < 0.05 as compared to scrambled controls in 3 independent experiments. (<b>E</b>)–(<b>F</b>), 3T3 fibroblasts were transiently transfected by siRNAs to Duox1 or Duox2 and analyzed for expression of mRNA in 3 independent experiments (<b>E</b>) and Nox4 and Duox1/2 proteins in 2 experiments (<b>F</b>). The mRNA expression levels were normalized to those in cells treated with non-targeting (NT) siRNA; (*) p < 0.05 as compared to NT controls. The western blots are typical of 2 experiments. (<b>G</b>)–(<b>H</b>), MSC were transiently transfected by siRNAs to Nox4, Duox1 or Duox2, and analyzed for mRNA in 3 independent experiments (<b>G</b>), and Nox4 protein expression in 2 experiments (<b>H</b>). The mRNA expression levels were normalized to those in NT controls; (*) p < 0.05 as compared to the NT controls. In this case Duox1/2 protein expression was not significantly altered by corresponding siRNAs (data not shown).</p