![]() ![]() Data were representative of two (A, B and C) or three (E, F and G) independent experimentsįig. 01 (two-tailed unpaired Student's t test) (A and C). Data were means +- SEM from three biological replicates, *represents P <. (G) Upper picture: immunofluorescence detection of Cyp26a1 expression in CD45 + CD3 - CD122 + NK cells isolated from the uterus on gd5 (63 x objective) lower picture: confocal photography of Cyp26a1 + NK cells and Cyp26a1 - NK cells isolated from the uterus on gd5 (20 x and 63 x objective). (F) Percentages of Cyp26a1 + NK cells in gated CD45 + CD3 - CD122 + NK and CD122 + NK cells in gated CD45 + CD3 - cells in the uterus on gd4-gd7. (E) Representative graph of flow cytometric assays to detect CD45 + CD3 - CD122 + Cyp26a1 + NK cells in the uterus on gd4-gd7, the marker molecules were as follows: CD3-PE, CD45-PerCP-Cy5.5, CD122-APC and Cyp26a1-Alexa Fluor 488, The cells under gate CD45 + CD3 - shown in the figure. (D) qPCR to detect the expression of Cyp26a1 in CD45 + CD3 - CD122 + NK cells isolated from the uterus on gd4-gd7. (C) qPCR to detect the expression of Cyp26a1 in CD45 + CD3 - CD122 + NK cells isolated from the uterus and spleen on gd5. (B) Western blotting to detect the expression of Cyp26a1 on gd4-gd7 uterine tissue. (A) qPCR to detect the expression of Cyp26a1 on gd4-gd7 uterine tissue. All data are presented as mean +- SD.ġ FIGURE Expression of Cyp26a1 in mouse uterine tissue and uterine NK cells during the peri-implantation period. The percentage of CD45 + cells in total bone marrow cells (center) and CD45 + GFP + cells in whole GFP + fraction (right). ( C ) Flow cytometry analysis of Control and Col2a1-CXCL12 cKO bone marrow cells. Green = Cxcl12-GFP red = tdTomato magenta = CD45-Alexa647 gray = DAPI. Dotted area = magnified view of Cxcl12 -GFP + reticular stromal cells and neighboring CD45 + hematopoietic cells. ( B ) CD45 staining of Control (left panels) and Col2a1-CXCL12 cKO (right panels) bone marrow. In Col2a1-CXCL12 cKO mice, Cxcl12-GFP + cells lose functional CXCL12 expression but still express GFP in a Cxcl12 promoter/enhancer-dependent manner. Col2a1-cre Cxcl12 GFP/+ R26R tdTomato mice were mated with Cxcl12 fl/fl mice to generate Col2a1-cre Cxcl12 GFP/fl R26R tdTomato (Col2a1-CXCL12 cKO) and Cxcl12 GFP/fl R26R tdTomato (Control) mice. ( A ) Conceptual diagram of CXCL12 conditional deletion experiment. Statistical significance was determined by unpaired two-tailed Student's t -test: * p < 0.05 ** p < 0.01 *** p < 0.001.ģ Fig CXCL12 is functionally important for maintaining stromal-hematopoietic coupling. ![]() Similar observations were achieved in other two experiments. Shown are dot plot graphs from one of three individual analyses. ( D ) Apoptosis of cells in WT and trappc9-deficient (KO) ASC cultures was examined by flow cytometry-based analysis of phosphatidylserine exposure (Annexin V-FITC staining) and plasma membrane integrity (propidium iodide staining). Images captured from 3 randomly chosen visual fields from cells for each genotype were used for cell counting. ( C ) Quantification of cells with two or more nuclei revealed that trappc9-null (KO) ASCs were defective in cell division. Cell counting was performed with a hemocytometer. ASCs were cultured at a density of 1 x 10 5 in T25 flasks and counted every 24 h for 6 days. ( B ) Comparison of the growth rate between WT and trappc9-deficient (KO) ASCs. Arrowheads point to cells containing two or more nuclei. ( A ) Confocal images showed that ASCs isolated from WT and trappc9-null (KO) mice expressed mesenchymal stem cell markers and were devoid of hematopoietic CD45. Both WT and KO ASCs at passage-3 were subjected to analyses. ASCs were isolated from WT and trappc9-deficient mice and cultured as in Methods. Compromised self-renewal of ASCs cultured from trappc9-null mice. ![]()
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