Journal: Brain : a journal of neurology

Article Title: Connexin43 mimetic peptide reduces vascular leak and retinal ganglion cell death following retinal ischaemia.

doi: 10.1093/brain/awr338

Figure Lengend Snippet: Figure 3 Three hours of hypoxia and 6 h of reperfusion lead to significant endothelial cell death in vitro in rat brain microvascular endothelial cells (RBMVEC; A). Non-specific gap junction blocker carbenoxolone, non-specific hemichannel blocker LaCl3, and connexin43 (Cx43) mimetic peptide protected endothelial cells against hypoxic injury, with the number of viable cells significantly higher than no treatment, while scrambled peptide did not have any protective effects. The number of viable cells was expressed as percentage of the control without hypoxia. Hypoxia and reperfusion also lead to significant propidium iodide dye uptake into primary rat brain microvascular endothelial cells indicating open hemichannels (B). Carbenoxolone, LaCl3 and connexin43 mimetic peptide significantly prevented dye uptake compared to no treatment, indicating hemichannel closure, while scrambled peptide did not have any effect. Stars denote statistical significance when compared to the control group or compared between groups in brackets; P 5 0.05.

Article Snippet: Rat brain microvascular endothelial cells (R840K-05a, Cell Applications) were plated into 24-well plates (1 105 cells/well) in rat brain microvascular endothelial cell growth media (R819K-500, Cell Applications) and allowed to settle for 16 h. Medium was then removed and replaced with Dulbecco’s Modified Eagle’s Medium/F12 containing 0.5% foetal bovine serum and 1% glutamine.

Techniques: In Vitro, Control

Journal: Mucosal immunology

Article Title: Airway structural cells regulate TLR5-mediated mucosal adjuvant activity.

doi: 10.1038/mi.2013.66

Figure Lengend Snippet: Figure 4 Flagellin is rapidly and totally degraded in airways conducts. C57BL/6 mice (n¼ 3–6) were treated intravenously or intranasally (i.n.) with flagellin. Spleen (a) and lung (b) were sampled at 2 h and Il22 mRNA levels were determined by quantitative PCR. Levels of mRNA in mock animal were arbitrarily set to 1 and used to calculate relative gene expression. (c–g) Mice (n ¼ 3–4) were stimulated i.n. with 1mg (d, e, g) or 10mg (c, f) flagellin and lungs and bronchoalveolar lavage (BAL) of mock or flagellin-treated mice were sampled at indicated time. " " and "þ " indicate BAL from mock animals and BAL supplemented ex vivo with flagellin, respectively. In panels c–e experiments were conducted on NMRI mice, whereas Tlr5 / and C57BL/6 (WT) animals were used for panels (f, g). (c) Kinetic of flagellin degradation. Flagellin (upper panel) or endogenous IgG (lower panel) were analyzed by SDS–polyacrylamide gel electrophoresis and immunoblot. For each time, the immunoblot for two animals were shown. (d) Concentration of flagellinwasestimated byELISA.(e)Timecourse analysisofflagellinactivity in sampled BAL. Epithelial cells Caco-2 were stimulated 24h with BAL isolated from flagellin-treated mice sampling at indicated time or medium as control ( ). Levels of IL-8 in supernatants were determined by ELISA. Resultsareexpressedasmean±s.d.Statisticalsignificance(*Po0.05)was assessed by Mann–Whitney test compared with phosphate-buffered saline (a, b) or t0 (e) group. (f) Flagellin degradation in BAL 2h after nasal administration was analyzed by flagellin-specific immunoblot. For each group, the immunoblot for 3 animals were shown. (g) Concentration of flagellin was estimated by ELISA at 2 and 6 h. ND, not done.

Article Snippet: The normal human bronchial epithelial cell BEAS-2B was grown in complete epithelial cell culture medium (Promocell GmbH, Heidelberg, Germany).

Techniques: Real-time Polymerase Chain Reaction, Gene Expression, Ex Vivo, Polyacrylamide Gel Electrophoresis, Western Blot, Concentration Assay, Isolation, Sampling, Control, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Saline

Journal: Mucosal immunology

Article Title: Airway structural cells regulate TLR5-mediated mucosal adjuvant activity.

doi: 10.1038/mi.2013.66

Figure Lengend Snippet: Figure 7 Airway epithelial cells replicate the lung signature to flagellin (a, b) Transcripts analysis of microdissected lung compartments. C57BL/6 mice (n ¼ 2–3) were treated i.n. with phosphate-buffered saline or flagellin 2 h. Bronchoalveolar lavage (BAL) and lungs were sampled. Lungs were prepared for laser microdissection of bronchial epithelium and alveolar compartment. Level of Ccl20 mRNA (a) and others proinflammatory genes transcripts (b) in bronchial epithelium and whole alveolar compartments, BAL cells or total lung were determined by quantitative PCR (qPCR). (c, d) Activation of human epithelial cells by flagellin treatment. Normal bronchial epithelial cell line BEAS-2B were stimulated 1 h with flagellin (c). Bronchial and alveolar epithelial cell line, 16HBE and A549, respectively, were stimulated 2 h with flagellin (d). Total RNA was extracted and proinflammatory gene expression analyzed by qPCR. Levels of mRNA in mock animals or cells were arbitrarily set to 1 and used to calculate relative gene expression of flagellin-treated animals or cells. Results are expressed as mean±s.d. Statistical significance (*Po0.05) was assessed by Mann–Whitney test.

Article Snippet: The normal human bronchial epithelial cell BEAS-2B was grown in complete epithelial cell culture medium (Promocell GmbH, Heidelberg, Germany).

Techniques: Saline, Laser Capture Microdissection, Real-time Polymerase Chain Reaction, Activation Assay, Gene Expression, MANN-WHITNEY

Journal: Nature cell biology

Article Title: A complex secretory program orchestrated by the inflammasome controls paracrine senescence

doi: 10.1038/ncb2784

Figure Lengend Snippet: (a) Co-culture with cells undergoing OIS induces the arrest of normal IMR90 cells. Normal IMR90 human fibroblasts expressing the fluorescent marker mCherry (IMR90 Cherry), were mixed with IMR90 ER:RAS cells. When indicated 200 nM 4OHT was added to activate ER:RAS. Growth curves represent the number of IMR90 ER:RAS (left) or IMR90 Cherry cells (right) present in the co-cultures. Data is a representative experiment of n=2 independent experiments for days 0-7 and mean +/− s.d. of n= 3 independent experiments for day 8. The source data for 2 independent experiments and statistics for day 8 (Student’s t-test) are provided in . (b) Co-culture with IMR90 MEK:ER cells induces the arrest of normal IMR90 cells. The percentage of positive BrdU cells for each population in the cocultures 4 days after 4OHT induction is shown. Data is a representative experiment. The source data for 2 independent experiments are provided in . (c) IMR90 ER:RAS or IMR90 ER cells were co-cultured with normal IMR90 Cherry fibroblasts. BrdU incorporation at day 7 shows that co-culture with IMR90 ER:RAS but not with IMR90 ER induces the arrest of normal IMR90 Cherry cells (centre). Data is a representative experiment. The source data for 2 independent experiments are provided in . Representative images are shown (right). Scale bar, 30 μm. (d) Normal HMEC or IMR90 cells suffer growth arrest when co-cultured with HMEC cells undergoing OIS. HMEC-hTERT (centre) or IMR90 (right) cells expressing Cherry as a fluorescent marker were co-cultured with HMEC-TERT ER:RAS or HMEC-TERT vector cells in the presence of 100 nM 4OHT. Growth curves showing growth arrest of HMEC-TERT Cherry (centre) or IMR90-Cherry cells (right) are shown. Data is a representative experiment.

Article Snippet: HMEC-hTert cells were cultured in mammary epithelial cell growth media (PromoCell).

Techniques: Co-Culture Assay, Expressing, Marker, Cell Culture, BrdU Incorporation Assay, Plasmid Preparation