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: Molecular Metabolism

Article Title: Activated macrophages control human adipocyte mitochondrial bioenergetics via secreted factors

doi: 10.1016/j.molmet.2017.07.008

Figure Lengend Snippet: Effects of LPS/IFNγ- and IL10/TGFβ-activated primary MΦ-CM on bioenergetics of human SGBS and primary adipocytes . Primary human CD14 + monocytes from 4 healthy donors were cultured, differentiated to macrophages (naïve MΦs) and activated with LPS/IFNγ or IL10/TGFβ as described in . (A) Morphological changes detected by bright field microscopy (one representative experiment shown). (B) mRNA expression of CD163 and CD40 analyzed with ΔΔCT method using naïve MΦs as Calibrator (n = 4). (C) CD163 and CD40 mRNA expression presented as ratio. (B, C) *p < 0.05 vs. LPS/IFNγ-activated MΦ, ***p < 0.001 vs. LPS/IFNγ-activated MΦ, ###p < 0.001 vs. naïve MΦs. (D) mRNA expression of UQCRC2 and NDUFB8 analyzed with ΔΔCT method using naïve THP1 macrophages as Calibrator (n = 4). ( E–G) SGBS adipocytes were treated with either cell-free control media, LPS/IFNγ-activated or IL10/TGFβ-activated MΦ-CM collected from 3 different donors for 48 h as described in and analyzed as described in Figure 4 , Figure 5 . Data are normalized to cell number (DNA content) and are the mean + SEM of 5 independent experiments each performed in 2–8 wells condition. OCR and ECAR traces vs time are shown in ( Figure S4B and C ). (E) ATP-linked respiration of SGBS adipocytes after treatment for 48 h with indicated MΦ-CM. (F) ATP-linked respiration of SGBS adipocytes presented as relative change in OCR in percent of cell-free control. (G) ECARs due to glycolysis of SGBS adipocytes after treatment for 48 h with indicated MΦ-CM. (H – J) Human primary subcutaneous adipocytes were treated with either cell-free control media, LPS/IFNγ-activated or IL10/TGFβ-activated MΦ-CM from 3 different donors as described in . Bioenergetic profile was analyzed as described in Figure 4 , Figure 5 . Data are presented as fold change to the mean rates of cell-free control for each donor and are the mean + SEM of experiments from 3 adipocyte donors performed in 6–8 wells per condition and donor. OCR and ECAR traces vs time are shown in ( Figure S4E and F ) (H) ATP-linked respiration of human primary adipocytes after treatment for 48 h with indicated MΦ-CM. (I) ATP-linked respiration of human primary adipocytes presented as relative change in OCR in percent of cell-free control. (J) ECARs due to glycolysis of human primary adipocytes after treatment for 48 h with indicated MΦ-CM. (E)–(J) *p < 0.05 vs. LPS/IFNγ-activated MΦ-CM; **p < 0.01 vs. LPS/IFNγ-activated MΦ-CM; ***p < 0.001 vs. LPS/IFNγ-activated MΦ-CM; #p < 0.05 vs. cell-free control media.

Article Snippet: Human CD14 + blood monocytes from 4 healthy donors were purchased from Zenbio (Research Triangle Park, NC, USA) and differentiated with 10 ng/ml CSF1 for 7 days to adherent macrophages (MΦ) before same stimulation as for THP1 cells was performed.

Techniques: Cell Culture, Microscopy, Expressing, Control