Figure 1. Accelerated HSC decline and myeloproliferation in miR-146a–deficient mice during chronic inflammation.

(A) MiR-146a and miR-146b expression in FACS-sorted HSPC populations by Taqman RT-qPCR. Lin-BM, lineage negative bone marrow cells; L⁻S⁺K⁺ (LSK), Lin⁻Sca1⁺cKit⁺; HSC, LSK CD150⁺CD48⁻; L⁻S⁻K⁺, Lin⁻Sca1⁻cKit⁺; L⁻S⁺K⁻, Lin⁻Sca1⁺cKit⁻; miR-146a KO BM, total bone marrow cells from Mir146a^−/− mice. (B). Representative FACS plots of LSK cells and CD150⁺CD48⁻ or EPCR⁺ HSCs from BM of 8-month-old wild-type (WT) and miR-146a KO mice. Quantification of number of BM CD45⁺ cells, LSK cells, LSK CD150⁺CD48⁻ HSCs, LSK EPCR⁺ HSCs and percent of LSK CD150⁺CD48⁻ HSCs of total BM from 8-month-old (C) and 12-month-old (D) WT and miR-146a KO mice by FACS. (E) Quantification of percent of LSK cells of total BM and percent of HSCs of LSK gate from BM of 8-month-old WT and miR-146a KO mice by FACS. (F) Quantification of number of LSK cells and LSK CD150⁺CD48⁻ HSCs from spleen of 8-month-old WT and miR-146a KO mice by FACS. (G). Total number of LSK CD150⁺CD48⁻ or LSK CD150⁺CD48⁻ HSCs from BM and spleen of 6-month-old WT and miR-146a KO mice. (H)–(J) 8-Week-old WT and miR-146a KO (miR KO) mice were subjected to repeated low-dose of intraperitoneal LPS stimulation (1 mg LPS/kg of body weight for 8 times) or PBS control spread over a month. At the end the month, four groups of mice were harvested for FACS analysis. (H) Quantification of percent of LSK cells of total BM, CD150⁺CD48⁻ HSCs of LSK gate, and number of CD11b⁺ myeloid cells in BM. I. Number of myeloid cells (CD11b⁺ or Gr1⁺) in peripheral blood. (J) Quantification of total number of LSK CD150⁺CD48⁻ HSCs, LSK EPCR⁺ HSCs and LSK cells in spleen.

DOI: http://dx.doi.org/10.7554/eLife.00537.003

Figure 1—figure supplement 1. HSPC FACS analysis and colony-forming ability in 6-week-old and 4-month-old WT and miR-146a KO mice.

(A) Quantification of number of white blood cells (CD45), T cells (CD3ε), myeloid cells (CD11b), B cells (CD19), nucleated erythrocytes (Ter119), LSK cells, LSK CD150⁺CD48⁻ HSCs and LSK CD150⁺CD48⁻EPCR⁺ HSCs from spleen of 6-week-old WT and miR-146a KO mice by FACS. (B) Quantification of number of white blood cells (CD45), LSK cells, LSK CD150⁺CD48⁻ HSCs, Lin-cKit⁺Sca1⁻ myeloid progenitors from BM of 6-week-old WT and miR-146a KO mice by FACS. (C) Colony forming units (CFU) in vitro in methylcellulose medium per 100,000 total BM cells from 6-week-old WT and miR-146a KO mice. (D) Quantification of number or percent of white blood cells (CD45), T cells (CD3ε), myeloid cells (CD11b), B cells (CD19), LSK cells and LSK CD150⁺CD48⁻ HSCs from BM of 4-month-old WT and miR-146a KO mice by FACS. (E) (related to Figure 1H–J) 8-Week-old WT and miR-146a KO (miR KO) mice were subjected to repeated low-dose of intraperitoneal LPS stimulation (1 mg LPS/kg of body weight for 8 times) spread over a month. WT and miR KO mice receiving phosphate-buffered saline (PBS) injection were included as controls. At the end the month, four groups of mice (WT PBS, miR KO PBS, WT LPS, and miR KO LPS) were harvested for FACS analysis. Spleen weight, number of CD45⁺, CD11b⁺, and Ter119⁺ cells in spleen were shown.

DOI: http://dx.doi.org/10.7554/eLife.00537.004