ets2 floxed mice  (Jackson Laboratory)

Journal: Aging (Albany NY)

Article Title: Ets2 suppresses inflammatory cytokines through MAPK/NF-κB signaling and directly binds to the IL-6 promoter in macrophages

doi: 10.18632/aging.102480

Figure Lengend Snippet: LPS and VSV promote Ets2 expression and nuclear translocation. ( A , B ) Ets2 mRNA expression in mouse primary peritoneal macrophages stimulated with 100 ng/ml LPS ( A ) or VSV at an MOI of 10 ( B ) for the indicated times, as detected by real-time PCR (n=3). ( C ) Immunoblot analysis and quantification of Ets2 expression in mouse primary peritoneal macrophages stimulated with 100 ng/ml LPS or VSV at an MOI of 10 for the indicated times. ( D , E ) Immunoblot analysis ( D ) and quantitative measurement ( E ) of nuclear/total lysate ratio of Ets2 of mouse primary peritoneal macrophages pretreated with SB203580, PD98059, or SP600125 (20 μM for 30 min) and then stimulated with or without 100 ng/ml LPS for 3 h. U6 was used as an internal reference for the nucleus. Data are shown as the mean ± s.d. Student’s t-test compared with the control group. *, P<0.05, and **, P<0.01.

Article Snippet: Ets2 flox mice were purchased from the Jackson Laboratory and mated with Lyz2-Cre mice.

Techniques: Expressing, Translocation Assay, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Aging (Albany NY)

Article Title: Ets2 suppresses inflammatory cytokines through MAPK/NF-κB signaling and directly binds to the IL-6 promoter in macrophages

doi: 10.18632/aging.102480

Figure Lengend Snippet: Ets2 inhibits LPS-induced IL-6 and TNF-α production in macrophages. ( A ) Immunoblot analysis of the expression of Ets2 in mouse primary peritoneal macrophages transfected with control siRNA (Ctrl) or Ets2-targeted siRNA (Ets2) with LPS stimulation for indicated time. ( B, C ) IL-6 ( B ) and TNF-α ( C ) mRNA expression of cells stimulated with 100 ng/ml LPS for the indicated times. ( D , E ) IL-6 ( D ) and TNF-α ( E ) mRNA expression in Ets2 fl/fl Lyz2cre − or Ets2 fl/fl Lyz2cre + mouse primary peritoneal macrophages stimulated with 100 ng/ml LPS for the indicated times. ( F , G ) ELISA assay of IL-6 ( F ) and TNF-α ( G ) in the supernatant of the cells stimulated with 100 ng/ml LPS for the indicated times. Data are shown as the mean ± s.d. of three samples. Student’s t-test compared with the control or Ets2 fl/fl Lyz2cre - group. *, P<0.05, and **, P<0.01.

Article Snippet: Ets2 flox mice were purchased from the Jackson Laboratory and mated with Lyz2-Cre mice.

Techniques: Western Blot, Expressing, Transfection, Control, Enzyme-linked Immunosorbent Assay

Journal: Aging (Albany NY)

Article Title: Ets2 suppresses inflammatory cytokines through MAPK/NF-κB signaling and directly binds to the IL-6 promoter in macrophages

doi: 10.18632/aging.102480

Figure Lengend Snippet: Ets2 inhibits VSV-induced IFN-β, IL-6, and TNF-α production in macrophages. ( A – C ) IFN-β ( A ), IL-6 ( B ) and TNF-α ( C ) mRNA expression of cells stimulated with VSV at an MOI of 10 for the indicated times. d-f: IFN-β ( D ), IL-6 ( E ), and TNF-α ( F ) mRNA expression in Ets2 fl/fl Lyz2cre − or Ets2 fl/fl Lyz2cre + mouse primary peritoneal macrophages stimulated with VSV at an MOI of 10 for the indicated times. ( G – I ) ELISA assay of IFN-β ( G ), IL-6 ( H ), and TNF-α ( I ) in the supernatants of cells stimulated with VSV at an MOI of 10 for the indicated times. ( J – L ) ELISA assay of IFN-β ( J ), IL-6 ( K ), and TNF-α ( L ) in the supernatants of the cells stimulated with VSV at an MOI of 10 for the indicated times in Ets2 fl/fl Lyz2cre − or Ets2 fl/fl Lyz2cre + mouse primary peritoneal macrophages. Data are shown as the mean ± s.d. of three samples. Student’s t-test compared with the control or Ets2 fl/fl Lyz2cre - group. *, P<0.05, and **, P<0.01.

Article Snippet: Ets2 flox mice were purchased from the Jackson Laboratory and mated with Lyz2-Cre mice.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Control

Journal: Aging (Albany NY)

Article Title: Ets2 suppresses inflammatory cytokines through MAPK/NF-κB signaling and directly binds to the IL-6 promoter in macrophages

doi: 10.18632/aging.102480

Figure Lengend Snippet: Ets2 inhibits LPS-induced IL-6 and TNF-α production and improves the survival of CLP-induced sepsis in vivo . ( A , B ) ELISA assay of IL-6 ( A ) and TNF-α ( B ) in the serum of Ets2 fl/fl Lyz2cre − or Ets2 fl/fl Lyz2cre + mice, intraperitoneally injected with LPS (1 μg/g body weight) for 5 h (n=3 per phenotype). ( C , D ) ELISA assay of IL-6 ( C ) and TNF-α ( D ) in the serum of Ets2 fl/fl Lyz2cre − or Ets2 fl/fl Lyz2cre + mice at 6 h after CLP surgery (n=4 per phenotype). ( E – G ) Continuous IL-6 ( E ) and TNF-α ( F ) at postoperative day 1, 3, 5, 7, and survival rate ( G ) of Ets2 fl/fl Lyz2cre − or Ets2 fl/fl Lyz2cre + mice given CLP or Sham surgery (n=10 per phenotype). Data are shown as the mean ± s.e.m. Student’s t-test compared with the Ets2 fl/fl Lyz2cre - group for ELISA experiments. A log-rank test was used for survival data. *, P<0.05.

Article Snippet: Ets2 flox mice were purchased from the Jackson Laboratory and mated with Lyz2-Cre mice.

Techniques: In Vivo, Enzyme-linked Immunosorbent Assay, Injection

Journal: Aging (Albany NY)

Article Title: Ets2 suppresses inflammatory cytokines through MAPK/NF-κB signaling and directly binds to the IL-6 promoter in macrophages

doi: 10.18632/aging.102480

Figure Lengend Snippet: Ets2 inhibits NF-κB and MAPK signaling. ( A , B ) Immunoblot analysis of phosphorylated and total ERK1/2, JNK, p38 and NF-κB p65 in mouse primary peritoneal macrophages transfected with control siRNA (Ctrl siRNA) or Ets2-targeted siRNA (Ets2 siRNA) ( A ) and primary peritoneal macrophages of Ets2 fl/fl Lyz2cre − or Ets2 fl/fl Lyz2cre + mice ( B ), stimulated with 100 ng/ml LPS for the indicated times. ( C , D ) Immunoblot analysis of phosphorylated and total ERK1/2, JNK, p38, and NF-κB p65 in primary peritoneal macrophages transfected with control siRNA (Ctrl siRNA) or Ets2-targeted siRNA (Ets2 siRNA) ( C ) and primary peritoneal macrophages of Ets2 fl/fl Lyz2cre − or Ets2 fl/fl Lyz2cre + mice ( D ) stimulated with VSV at an MOI of 10 for the indicated times.

Article Snippet: Ets2 flox mice were purchased from the Jackson Laboratory and mated with Lyz2-Cre mice.

Techniques: Western Blot, Transfection, Control

Journal: Aging (Albany NY)

Article Title: Ets2 suppresses inflammatory cytokines through MAPK/NF-κB signaling and directly binds to the IL-6 promoter in macrophages

doi: 10.18632/aging.102480

Figure Lengend Snippet: Ets2 inhibits IL-6 transcription by binding to the IL-6 promoter. ( A ) ChIP analysis of the IL-6 promoter using an Ets2 antibody in mouse primary peritoneal macrophages treated with LPS for the indicated times. ( B ) Luciferase assay of IL-6 reporter gene expression in HEK293 cells transfected with 50 ng of a MyD88-expressing plasmid, 90 ng of an IL-6 luciferase reporter plasmid, and 10 ng of a pTK-Renilla-luciferase reporter plasmid, together with 0, 10, 25 or 50 ng of an Ets2-expressing plasmid. Luciferase activity was measured and normalized to Renilla luciferase activity. Data are shown as the mean ± s.d. Student’s t-test compared with the control group. **, P<0.01.

Article Snippet: Ets2 flox mice were purchased from the Jackson Laboratory and mated with Lyz2-Cre mice.

Techniques: Binding Assay, Luciferase, Gene Expression, Transfection, Expressing, Plasmid Preparation, Activity Assay, Control

Journal: Aging (Albany NY)

Article Title: Ets2 suppresses inflammatory cytokines through MAPK/NF-κB signaling and directly binds to the IL-6 promoter in macrophages

doi: 10.18632/aging.102480

Figure Lengend Snippet: Ets2 inhibits NF-κB-dependent IL-6 transcription. ( A ) Schematic diagram of truncated IL-6 promoter-reporter plasmids constructed by deleting the regions at −1176/−1081, −1176/−801, −1176/−451, and −1176/−171. ( B ) Luciferase assay in HEK293 cells transfected with 50 ng of a MyD88-expressing plasmid, 90 ng of the truncated IL-6 promoter-luciferase reporter plasmids, and 10 ng of a pTK-Renilla-luciferase reporter plasmid, together with 0, 10, 25 or 50 ng of an Ets2-expressing plasmid. Luciferase activity was measured and normalized by Renilla luciferase activity. ( C ) IL-6 promoter sequence shown from position -80 to -21. ( D ) Luciferase assay in HEK293 cells transfected with 50 ng of a p65-expressing plasmid, 90 ng of the truncated IL-6 luciferase reporter plasmids, and 10 ng of a pTK-Renilla-luciferase reporter plasmid, together with 0, 10, 25 or 50 ng of an Ets2-expressing plasmid. Luciferase activity was measured and normalized to Renilla luciferase activity. Data are shown as the mean ± s.d. Student’s t-test compared with the control group. **, P<0.01.

Article Snippet: Ets2 flox mice were purchased from the Jackson Laboratory and mated with Lyz2-Cre mice.

Techniques: Construct, Luciferase, Transfection, Expressing, Plasmid Preparation, Activity Assay, Sequencing, Control