Journal: Aging Cell

Article Title: The activation of cGAS‐STING pathway causes abnormal uterine receptivity in aged mice

doi: 10.1111/acel.14303

Figure Lengend Snippet: CD14 protein levels and its effects on uterine receptivity in aged mice. (a) Proteomic analysis of young (D4) and aged mouse (AGED D4) uteri on day 4 of pregnancy. (b) Western blot analysis of CD14 protein levels in young and aged mouse uteri on day 4 of pregnancy. (c) CD68 and CD14 immunofluorescence in young and aged mouse uteri on day 4 of pregnancy. Scale bar, 50 μm. (d) Uterine CD14 concentration after ovariectomized mice were injected subcutaneously with 0, 3, 10, or 25 ng E 2 for 7 days. (e) CD14 concentration in the cultured medium after Progerin gene was overexpressed in mouse epithelial cells. Con, empty vector control; Progerin , Progerin overexpression. (f) CD14 concentration in the cultured medium after mouse epithelial cells were transfected with SDNA for 48 h. (g) CD14 concentration in the cultured medium after mouse epithelial cells were treated with ADU S100 for 48 h. (h) Immunofluorescence of the receptivity‐related proteins after mouse epithelial organoids were treated with CD14 for 48 h. Scale bar, 100 μm. (i) Western blot analysis of the receptivity‐related proteins after mouse epithelial organoids were treated with CD14 for 48 h. (j) A representative photograph showing the number of implantation sites on day 5 after recombinant CD14 was injected into the uterine lumen on day 4 of pregnancy. (k) Statistical analysis on the number of implantation sites on day 5 after recombinant CD14 was injected into the uterine lumen on day 4 of pregnancy. * p < 0.05; **, p < 0.01; *** p < 0.001.

Article Snippet: Luminal epithelial cells were treated with ADU‐S100 (2 and 20 μM, HY‐12885A, MedChemExpress), Salmon SDNA (0.2 and 2 μg/mL) or CD14 (0.1 and 1 μg/mL, HY‐P75444, MedChemExpress) in DMEM/F12 with 2% charcoal‐treated FBS (cFBS, Biological Industries, Cromwell, CT).

Techniques: Western Blot, Immunofluorescence, Concentration Assay, Injection, Cell Culture, Plasmid Preparation, Control, Over Expression, Transfection, Recombinant

Journal: Aging Cell

Article Title: The activation of cGAS‐STING pathway causes abnormal uterine receptivity in aged mice

doi: 10.1111/acel.14303

Figure Lengend Snippet: Effects of STING inhibitor (C176) on uterine receptivity and cGAS‐STING pathway. (a) Uterine CD14 protein levels young mice (D4), aged mice (AGED D4), and C176‐treated mice on day 4 of pregnancy. (b)CD14 immunofluorescence in young mice, aged mice, and C176‐treated mice on day 4 of pregnancy. Scale bar, 50 μm.(c) CD14 concentration in uteri of aged mice and C176‐treated mice. (d) CD14 concentration in serum of aged mice and C176‐treated mice. (e)Immunofluorescence of uterine AREG, MUC1 and MSX1 in young mice, aged mice, and C176‐treated mice. Scale bar, 50 μm. (f) Cytoplasmic dsDNA concentration in the uterus of young mice, aged mice, and C176‐treated mice. (g) Western blot analysis of the cGAS‐STING related proteins and receptivity‐related proteins in young mice, aged mice, and C176‐treated mice. (h) The protein levels CGAS‐STING pathway and uterine receptivity marker in mice on the D3 of pregnancy were injected with E2 (25 ng) and TLR4 inhibitor Resatorvid (10 mg/mL) subcutaneously for 24 h. (i) The protein levels CGAS‐STING pathway and uterine receptivity marker after epithelial organoids were overexpression of PROGERIN for 48 hr in the absence or presence of CD14 antibody. con, empty vector control; Progerin , Progerin overexpression. *, p < 0.05; **, p < 0.01; ****, p < 0.0001.

Article Snippet: Luminal epithelial cells were treated with ADU‐S100 (2 and 20 μM, HY‐12885A, MedChemExpress), Salmon SDNA (0.2 and 2 μg/mL) or CD14 (0.1 and 1 μg/mL, HY‐P75444, MedChemExpress) in DMEM/F12 with 2% charcoal‐treated FBS (cFBS, Biological Industries, Cromwell, CT).

Techniques: Immunofluorescence, Concentration Assay, Western Blot, Marker, Injection, Over Expression, Plasmid Preparation, Control

Journal: The EMBO Journal

Article Title: A G1‐like state allows HIV ‐1 to bypass SAMHD 1 restriction in macrophages

doi: 10.15252/embj.201696025

Figure Lengend Snippet: A, B Stimulated or unstimulated MDM were stained for classical macrophage markers (CD68, CD14) and M1 and M2 macrophage markers (CD163, CD80, CD86 and CD40) and analysed by FACS.

Article Snippet: Antibodies used were as follows: anti‐cdc2 (Cell Signaling Technology, Beverly, MA, USA); anti‐CDK2 (H‐298, Santa Cruz Biotechnology); anti‐pCDK2(Thr160) (Bioss Inc., MA, USA); anti‐CDK4 (DCS156, Cell Signaling Technology); anti‐CDK6 (B‐10, Santa Cruz Biotechnology); anti‐SAMHD1 (ab67820, Abcam, UK), beta‐actin (ab6276, abcam, UK); mouse anti‐MCM2 (BM‐28, BD Biosciences, UK); and rabbit anti‐MCM2 (SP85) from Sigma; pSAMHD1 (a kind gift from M. Benkirane) and ProSci (Poway, CA, USA); anti‐Geminin (NCL‐L‐Geminin, Leica); anti‐mouse F4/80 and CD11b (kind gift from S. Yona, UCL); anti‐human CD68, CD14, CD163, CD80, CD86, CD40 (kind gift from M. Noursadeghi).

Techniques: Staining

Journal: PLoS ONE

Article Title: MFHAS1 Is Associated with Sepsis and Stimulates TLR2/NF-κB Signaling Pathway Following Negative Regulation

doi: 10.1371/journal.pone.0143662

Figure Lengend Snippet: (A) HEK 293 cells and 293-MFHAS1 cells were transiently transfected with 100 ng TLR2 or 100 ng TLR2/50 ng CD14 expression plasmids, and 100 ng NF-κB-dependent luciferase reporter plasmid as well as 10 ng renilla plasmid. Post transfection for 24 h, these transfected cells were exposed to mock treatment, Pam3CSK4 100 ng/mL or 10 μg/mL for 6 h, and fold increase in luciferase activity was measured for NF-κB activation using dual luciferase kits. The relative luciferase activity was calculated from the ratio of NF-κB-Luc (firefly) activity to renilla activity. (B) HEK 293 cells and 293-MFHAS1 cells were transiently transfected with 100 ng TLR2, and 24 h post-transfected cells were untreated or exposed to Pam3CSK4 100 ng/mL or 10μg/mL. After treatment for 6 h, IL-6 expression was assayed by quantitative RT-PCR and normalized to GAPDH. (C) HEK 293 cells and 293-MFHAS1 cells were transiently transfected with 100 ng TLR2, and 24 h post-transfected cells were untreated or exposed to Pam3CSK4 100 ng/mL. The cell supernatant was collected and the amounts of IL-6 were determined by ELISA at 6 h post-treatment. Values are the means ± SD from at least three independent experiments. * p < 0.05, ** p < 0.01, or *** p < 0.001.

Article Snippet: pGL4.32[ luc2P /NF-κB–RE/Hygro] and renilla vector reporter plasmid were purchased from Promega® (Madison, USA), REPO TM AP-1 vector reporter plasmid and IRF7-Gal4 fusion vectors were purchased from GenomeDitech® (Shanghai, China), expression vectors for human TLR2 and monocyte differentiation antigen CD14 (CD14) were purchased from Genechem® (Shanghai, China).

Techniques: Transfection, Expressing, Luciferase, Plasmid Preparation, Activity Assay, Activation Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: MFHAS1 Is Associated with Sepsis and Stimulates TLR2/NF-κB Signaling Pathway Following Negative Regulation

doi: 10.1371/journal.pone.0143662

Figure Lengend Snippet: (A, B) HEK 293 cells and 293-MFHAS1 cells were transiently transfected with 100 ng TLR2 or 100 ng TLR2/50 ng CD14 expression plasmids, an100 ng NF-κB luciferase reporter plasmid (A) or 20 ng AP-1 luciferase reporter plasmid (B) and 10 ng renilla plasmid. 24 h post-transfected cells were exposed to mock treatment, Pam3CSK4 100 ng/mL or 10μg/mL. At 24 h posttreatment, fold increase in luciferase activity was measured for NF-κB or AP-1 activation using dual luciferase kits. The relative luciferase activity was calculated from the ratio of NF-κB/AP-1 (firefly) activity to renilla activity. (C, D) HEK 293 cells and 293-MFHAS1 cells were transiently transfected with 100 ng TLR2 or 100 ng TLR2/50 ng CD14 expression plasmids, and 24 h post-transfected cells were untreated or exposed to Pam3CSK4 100 ng/mL. After 24 h and 36 h posttreatment, induction of IL-6 expression was assayed by quantitative RT-PCR and normalized to β-actin (C). Cell supernatant was collected and the amounts of IL-6 were determined by ELISA (D). Values are the means ± SD from at least three independent experiments. * p < 0.05, ** p < 0.01, or *** p < 0.001.

Article Snippet: pGL4.32[ luc2P /NF-κB–RE/Hygro] and renilla vector reporter plasmid were purchased from Promega® (Madison, USA), REPO TM AP-1 vector reporter plasmid and IRF7-Gal4 fusion vectors were purchased from GenomeDitech® (Shanghai, China), expression vectors for human TLR2 and monocyte differentiation antigen CD14 (CD14) were purchased from Genechem® (Shanghai, China).

Techniques: Transfection, Expressing, Luciferase, Plasmid Preparation, Activity Assay, Activation Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: MFHAS1 Is Associated with Sepsis and Stimulates TLR2/NF-κB Signaling Pathway Following Negative Regulation

doi: 10.1371/journal.pone.0143662

Figure Lengend Snippet: (A) HEK293 and 293-MFHAS1 cells were transiently transfected with 100 ng TLR2 or 100 ng TLR2/50 ng CD14 expression plasmids, the pFR luciferase reporter gene along with plasmid expressing IRF7-Gal4 3 ng. Then these 24 h post-transfected cells were treated with mock, 100 ng/mL or 10μg/mL Pam3CSK4 for 24 h, and luciferase reporter gene activity was measured. (B) HEK293 and 293-MFHAS1 cells were transiently transfected with 100 ng TLR2 expression plasmids, and 24 h post-transfected cells were untreated or treated with 100 ng/mL Pam3CSK4 for 24 h. IFN-β mRNA expression was assayed by quantitative RT-PCR. (C) The relative IFN-β mRNA level was normalized to GAPDH. Values are the means ± SD from at least three independent experiments. * p < 0.05, ** p < 0.01, or *** p < 0.001.

Article Snippet: pGL4.32[ luc2P /NF-κB–RE/Hygro] and renilla vector reporter plasmid were purchased from Promega® (Madison, USA), REPO TM AP-1 vector reporter plasmid and IRF7-Gal4 fusion vectors were purchased from GenomeDitech® (Shanghai, China), expression vectors for human TLR2 and monocyte differentiation antigen CD14 (CD14) were purchased from Genechem® (Shanghai, China).

Techniques: Transfection, Expressing, Luciferase, Plasmid Preparation, Activity Assay, Quantitative RT-PCR