Journal: Aging Cell

Article Title: Activation of epidermal growth factor receptor signaling mediates cellular senescence induced by certain pro‐inflammatory cytokines

doi: 10.1111/acel.13145

Figure Lengend Snippet: Five pro‐inflammatory cytokines induce cellular senescence through EGFR activation. (a) The effect of cetuximab on senescence‐inducing pro‐inflammatory cytokines; the values of “Relative levels of SAHF‐ or β‐gal‐positive” were extracted from Table . The red box in the upper right corner indicates the cytokines inducing senescence under the indicated conditions. (b) The effect of gefitinib on senescence‐inducing pro‐inflammatory cytokines; the values of “Relative levels of SAHF‐ or β‐gal‐positive” were extracted from Table . (c) Protein levels of p‐EGFR (Tyr1068), EGFR, p‐Erk1/2 (Thr202/Tyr204), and Erk1/2 in cells with or without SDF‐1α treatment. (d) EGFR protein levels of IMR90 cells with or without EGFR knockdown. (e) Percentages of SAHF‐ or SA‐β‐gal‐positive cells in groups treated as indicated in the relevant figure. Data indicate the mean values calculated from three independent experiments (± SD ). The Western blots were performed at least three times and the representative pictures are presented

Article Snippet: The other reagents used are as follows: recombinant EGF, IL‐1β, IL‐7, IL‐13, IL‐15, GRO‐α, IL‐8, SDF‐1α, MCP‐3, MIP‐1α, MCP‐2, MIP‐3α, eotaxin‐3, bFGF, KGF, vEGF, IGF‐BP7, OPG, GM‐CSF (BBI Life Sciences, Shanghai, China); recombinant IL‐6, MMP‐3, TGF‐β1 (PeproTech); cetuximab (Merck KGaA); gefitinib, cisplatin, and LY3214996 (Selleck Chemicals Inc); MTT (3‐[4]‐2,5‐ diphenyltetrazolium bromide thiazolyl blue), X‐Gal (5‐bromo‐4‐chloro‐3‐indolyl‐β‐D‐galactopyranoside), DAPI (4′, 6‐diamidine‐2′‐phenylindole dihydrochloride), BrdU (5‐bromo‐2‐deoxyuridine), and PI (propidium iodide) (Sigma‐Aldrich); annexin V‐FITC/PI Apoptosis Detection Kit (Yeasen Biotech); Human IL‐8 Uncoated ELISA Kit (Thermo Scientific); Human Total MMP3 ELISA Kit and Human EGF ELISA Kit (Proteintech).

Techniques: Activation Assay, Knockdown, Western Blot

Journal: Aging Cell

Article Title: Activation of epidermal growth factor receptor signaling mediates cellular senescence induced by certain pro‐inflammatory cytokines

doi: 10.1111/acel.13145

Figure Lengend Snippet: Erk1/2 activation is necessary for EGF‐induced cellular senescence. (a) Cells were treated with or without 100 nM of EGF (for 5, 10, or 20 min), and the protein levels of p‐Erk1/2 and Erk1/2 were determined by Western blotting. (b) Protein levels of Erk1/2 in cells with or without Erk1/2 knockdown. (c–e) Statistical analysis of positive cells from BrdU (c), SAHF (d), or SA‐β‐gal staining (e) in the groups indicated in the figures. (f) the growth curves of the cells with indicated treatments. (g) Protein levels of p53, p21, p16, 53BP1, and γH2AX in above cells. (h) The statistical analysis of 53BP1 foci‐positive cells in the groups indicated in the figures. (i–k), Statistical analysis of positive cells from BrdU (i), SAHF (j), or SA‐β‐gal staining (k) in the groups indicated in the figures; LY3214996 (300 nM), a selective Erk1/2 inhibitor, was applied together with EGF (100 nM). (l), The growth curves of the cells with indicated treatments. Data indicate the mean values calculated from three independent experiments (± SD ). The Western blots were performed at least three times, and the representative pictures are presented

Article Snippet: The other reagents used are as follows: recombinant EGF, IL‐1β, IL‐7, IL‐13, IL‐15, GRO‐α, IL‐8, SDF‐1α, MCP‐3, MIP‐1α, MCP‐2, MIP‐3α, eotaxin‐3, bFGF, KGF, vEGF, IGF‐BP7, OPG, GM‐CSF (BBI Life Sciences, Shanghai, China); recombinant IL‐6, MMP‐3, TGF‐β1 (PeproTech); cetuximab (Merck KGaA); gefitinib, cisplatin, and LY3214996 (Selleck Chemicals Inc); MTT (3‐[4]‐2,5‐ diphenyltetrazolium bromide thiazolyl blue), X‐Gal (5‐bromo‐4‐chloro‐3‐indolyl‐β‐D‐galactopyranoside), DAPI (4′, 6‐diamidine‐2′‐phenylindole dihydrochloride), BrdU (5‐bromo‐2‐deoxyuridine), and PI (propidium iodide) (Sigma‐Aldrich); annexin V‐FITC/PI Apoptosis Detection Kit (Yeasen Biotech); Human IL‐8 Uncoated ELISA Kit (Thermo Scientific); Human Total MMP3 ELISA Kit and Human EGF ELISA Kit (Proteintech).

Techniques: Activation Assay, Western Blot, Knockdown, Staining

Journal: Aging Cell

Article Title: Activation of epidermal growth factor receptor signaling mediates cellular senescence induced by certain pro‐inflammatory cytokines

doi: 10.1111/acel.13145

Figure Lengend Snippet: Five pro‐inflammatory cytokines induce cellular senescence through EGFR activation. (a) The effect of cetuximab on senescence‐inducing pro‐inflammatory cytokines; the values of “Relative levels of SAHF‐ or β‐gal‐positive” were extracted from Table . The red box in the upper right corner indicates the cytokines inducing senescence under the indicated conditions. (b) The effect of gefitinib on senescence‐inducing pro‐inflammatory cytokines; the values of “Relative levels of SAHF‐ or β‐gal‐positive” were extracted from Table . (c) Protein levels of p‐EGFR (Tyr1068), EGFR, p‐Erk1/2 (Thr202/Tyr204), and Erk1/2 in cells with or without SDF‐1α treatment. (d) EGFR protein levels of IMR90 cells with or without EGFR knockdown. (e) Percentages of SAHF‐ or SA‐β‐gal‐positive cells in groups treated as indicated in the relevant figure. Data indicate the mean values calculated from three independent experiments (± SD ). The Western blots were performed at least three times and the representative pictures are presented

Article Snippet: The other reagents used are as follows: recombinant EGF, IL‐1β, IL‐7, IL‐13, IL‐15, GRO‐α, IL‐8, SDF‐1α, MCP‐3, MIP‐1α, MCP‐2, MIP‐3α, eotaxin‐3, bFGF, KGF, vEGF, IGF‐BP7, OPG, GM‐CSF (BBI Life Sciences, Shanghai, China); recombinant IL‐6, MMP‐3, TGF‐β1 (PeproTech); cetuximab (Merck KGaA); gefitinib, cisplatin, and LY3214996 (Selleck Chemicals Inc); MTT (3‐[4]‐2,5‐ diphenyltetrazolium bromide thiazolyl blue), X‐Gal (5‐bromo‐4‐chloro‐3‐indolyl‐β‐D‐galactopyranoside), DAPI (4′, 6‐diamidine‐2′‐phenylindole dihydrochloride), BrdU (5‐bromo‐2‐deoxyuridine), and PI (propidium iodide) (Sigma‐Aldrich); annexin V‐FITC/PI Apoptosis Detection Kit (Yeasen Biotech); Human IL‐8 Uncoated ELISA Kit (Thermo Scientific); Human Total MMP3 ELISA Kit and Human EGF ELISA Kit (Proteintech).

Techniques: Activation Assay, Knockdown, Western Blot

Journal: Applied and Environmental Microbiology

Article Title: Discovery of a Conjugative Megaplasmid in Bifidobacterium breve

doi: 10.1128/AEM.02871-14

Figure Lengend Snippet: Bacterial strains and plasmids used in this study a

Article Snippet: Recombinant E. coli cells containing pORI19 were selected on LB agar containing erythromycin and supplemented with X-Gal (5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside) at 40 μg ml −1 and 1 mM IPTG (isopropyl-β- d -galactopyranoside). table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain or plasmid Relevant feature(s) b Source or reference Strains Escherichia coli E. coli EC101 Cloning host, repA + Km r 53 Bifidobacterium breve JCM 7017 Isolate from human feces JCM JCM 7017-199 pORI19-tet-0199 insertion mutant of pMP7017 in JCM7017 This study UCC2003 Isolate from nursling stool 19 JCM 7019 Isolate from infant feces JCM NCFB 2258 Isolate from infant intestine NCFB UCC2003-TC B. breve UCC2003 transconjugant harboring pMP7017_199 This study JCM 7019-TC B. breve JCM7019 transconjugant harboring pMP7017_199 This study NCFB 2258-TC B. breve NCFB 2258 transconjugant harboring pMP7017_199 This study B. longum subsp. longum NCIMB 8809 Isolate from nursling stool NCIMB 1-6B Isolate from 6-year-old child 51 2-2B Isolate from 6-year-old child 51 44B Isolate from infant at 1 year 51 35B Isolate from infant at 1 year 51 NCIMB 8809-TC B. longum NCIMB 8809-TC transconjugant harboring pMP7017_199 This study Plasmids pAM5 pBC1-puC19-Tc r 54 pORI19 Em r ; Δ repA ; ori + , cloning vector 53 pORI19-tet-0199 Internal 937-bp fragment of pl_0199 and tetW cloned in pORI19 This study Open in a separate window a JCM, Japan Collection of Microorganisms; NCFB, National Collection of Food Bacteria; NCIMB, National Collection of Industrial and Marine Bacteria. b Em r , erythromycin resistance.

Techniques: Plasmid Preparation, Cloning, Mutagenesis, Clone Assay