Journal: Scientific Reports

Article Title: A PARP1-ERK2 synergism is required for the induction of LTP

doi: 10.1038/srep24950

Figure Lengend Snippet: ( a ) Recruited phosphorylated Erk2 and acetylated H4 (AcH4) to promoters of c-fos and zif268 in the chromatin of stimulated cerebral neurons (chromatin was crosslinked after stimulation; ChIP assay). Cerebral neurons of WT and PARP1-KO mice were stimulated by 3 repeats of 1 sec 100 Hz stimulation followed by 10 sec pause. Left: DNA segments in the promoters of c-fos and zif268 were amplified by RT-PCR after DNA isolation from crosslinked chromatin segments co-immunoprecipitated with phosphorylated Erk2 by antibody directed against the c-terminal of Erk2. Right: DNA segments in the promoters of c-fos and zif268 were amplified by RT-PCR after DNA isolation from crosslinked chromatin segments co-immunoprecipitated with antibody directed against acetylated histone H4 (AcH4; Methods). Each value represents the mean abundance of co-immunoprecipitated promoter fragments measured by 4 different reactions (with calculated variation coefficient) in 4 different experiments. ( b ) Proteins recovered from the crosslinked chromatin segments of stimulated cerebral neurons (3 repeats of 1 sec 100 Hz stimulation, 10 sec pause) of WT and PARP1 KO mice co-immunoprecipitated with PARP1 or Erk2 antibodies. The displayed results indicate: PARP1 binding to phosphorylated Erk2 in the stimulated cerebral neurons of WT mice. PARP inhibition improved their binding to histone H1, but impaired their binding to Elk1and CREB. Phosphorylated Erk2 scarcely bound to Elk1 and CREB in the chromatin of stimulated PARP1-KO cerebral neurons. Representative results of 4 different experiments.( c ) A schematic presentation of PARP1 dependent expression of immediate early gene, based on the results in panels ( a , b ) Binding of phosphorylated Erk2 to PARP1 induces its polyADP-ribosylation and release of its polyADP-ribosylated substrate, linker histone H1. This facilitates Erk-induced phosphorylation of transcription factor Elk1, hisone acetylation and gene expression.

Article Snippet: Antibodies directed against transcription factors c-Fos (Cell Signaling Technologies), Egr1 (Zif268; Cell Signaling Technologies), phosphorylated CREB (phosphorylation of serine-133; Cell Signaling Technologies) and Arc (Novous Biologicals, Cambridge, UK).

Techniques: Amplification, Reverse Transcription Polymerase Chain Reaction, DNA Extraction, Immunoprecipitation, Binding Assay, Inhibition, Expressing

Journal: PLoS ONE

Article Title: Th1-Induced CD106 Expression Mediates Leukocytes Adhesion on Synovial Fibroblasts from Juvenile Idiopathic Arthritis Patients

doi: 10.1371/journal.pone.0154422

Figure Lengend Snippet: SFbs from healthy donors were cultured in presence of medium (CTRL) or culture supernatants of unstimulated or anti-CD3/CD28 stimulated T helper cells of different phenotypes (classic and non-classic Th1 and Th17). A . SFbs vitality was evaluated by WST-1 assay after 24h and 48h of culture. Columns represent mean ± SE of % of viable SFbs compared to control condition (defined as 100% and shown as line in the Fig) in three experiments. Statistical analysis was performed by using the ANOVA test. B . After 48h SFbs morphology was evaluated by phase contrast microscope (magnification 200X). One representative experiment out of six is shown (one field out of five). Scale bar in all images = 100μm.

Article Snippet: Selected T cell clones with the following phenotypes: Th17 (CD161+IL-17+IFN-γ-), non-classic and classic Th1 (CD161+IL-17-IFN-γ+ and CD161-IL-17-IFN-γ+, respectively), were polyclonally stimulated with anti-CD3 plus anti-CD28 mAbs (human T Cell Activation/Expansion Kit, Miltenyi Biotec), for 72 hours to obtain culture conditioned supernatants, that were stored at -30°C.

Techniques: Cell Culture, WST-1 Assay, Control, Microscopy

Journal: PLoS ONE

Article Title: Th1-Induced CD106 Expression Mediates Leukocytes Adhesion on Synovial Fibroblasts from Juvenile Idiopathic Arthritis Patients

doi: 10.1371/journal.pone.0154422

Figure Lengend Snippet: SFbs from healthy donors were cultured for 48h in presence of medium alone (CTRL) or cultured supernatants of unstimulated (dark grey) or anti-CD3/CD28 stimulated (light grey) Th cells clones of different phenotypes (classic and non-classic Th1 and Th17) (A-B) or in the presence of TNF-α or IFN-γ cytokines or their combinations (D-E) in presence or absence of Etanercept (ETN) (G). Columns represent mean ± SE of % of CD106 positive SFbs (A) or of CD106 mean fluorescence intensity (MFI) (G) or both % of CD106 positive SFbs and CD106 mean fluorescence intensity (MFI) (D) of six different experiments. After 24h CD106 (B-E), TNF-αRβ (white) and IFN-γR2 (grey) (F) expression were evaluated by real time RT-PCR. Columns represents mean ± SE of mRNA expression (normalized on housekeeping gene mRNA and calculated as fold to the control, Ctrl) of three different experiments. * p < 0.05; ** p < 0.01, *** p < 0.001 stimulated condition versus ctrl or indicated by bar. IFN-γ (black), TNF-α (grey) and IL-17 (white) cytokine levels in anti-CD3/CD28 stimulated classic and non-classic Th1 and Th17-derived supernatants were evaluated by CBA flex set assay (C); columns represents mean ± SE of cytokine concentration (pg/ml) of five different supernatants for each Th phenotype.* p < 0.05 TNF-α versus IFN-γ or indicated by bar. Statistical analysis was performed by using the ANOVA test.

Article Snippet: Selected T cell clones with the following phenotypes: Th17 (CD161+IL-17+IFN-γ-), non-classic and classic Th1 (CD161+IL-17-IFN-γ+ and CD161-IL-17-IFN-γ+, respectively), were polyclonally stimulated with anti-CD3 plus anti-CD28 mAbs (human T Cell Activation/Expansion Kit, Miltenyi Biotec), for 72 hours to obtain culture conditioned supernatants, that were stored at -30°C.

Techniques: Cell Culture, Clone Assay, Fluorescence, Expressing, Quantitative RT-PCR, Control, Derivative Assay, Concentration Assay