Fig 1.
miR-181a1 and miR-181b1 expression in thymocytes, cTEC and mTECs in Mir181a1/b1fl/fl and FoxN1-Cre::Mir181a1/b1fl/fl mice.
Thymocytes, cTECs or mTECs were isolated using flow cytometry by staining with CD45, EpCAM, Ly51, UEA-1 and MHC II. Fig 1A. Thymocytes (CD45+) were sorted using a FacsAria. TECS (CD45-,EpCAM+) were isolated and then sorted into subpopulations: cTEC (UEA−1-Ly51+), mTEClo (UEA-1+Ly51−) mTEChi(UEA-1+Ly51-I-Ab) from both Mir181a1/b1fl/fl mice and Foxn1-Cre::Mir181a1/b1fl/fl. RNA was isolated and miR-181a1 and miR-181b1 was quantified using RT-PCR in the different populations. * = P<0.05, ** = P<0.01, *** = P<0.001.
Fig 2.
The effects of miR-181a1 and miR-181b1 loss on TEC distribution.
Fig 2A. Flow cytometric analysis of TEC (CD45−EpCAM+) subpopulations isolated in mice. The relative frequency of cTEC (UEA−1-Ly51+), mTEClo (UEA-1+Ly51−) mTEChi(UEA-1+Ly51+I-Ab+) are shown in both Mir181a1/b1fl/fl mice compared to Foxn1-Cre::Mir181a1/b1fl/fl. These are representative dot plots. This experiment was performed twice with at least 3 mice per group. Fig 2B. Flow cytometric analysis of TEC (CD45−EpCAM+) subpopulations isolated in mice. The relative frequency of cTEC (UEA−1-Ly51+), mTEClo (UEA-1+Ly51−) mTEChi(UEA-1+Ly51+I-Ab+) are shown as a percentage in the thymus in both Mir181a1/b1fl/fl mice compared to Foxn1-Cre::Mir181a1/b1fl/fl. This experiment was performed twice with at least 3 mice per group. Unpaired non-parametric t test was performed. * = P<0.05, ** = P<0.01.
Fig 3.
miR-181a1 and miR-181b1 does not affect TECs based on absolute numbers.
A. Flow cytometric analysis of TEC (CD45−EpCAM+MHC II+) subpopulations isolated in mice. The absolute number of total thymocytes, TEC, cTEC (UEA−1-Ly51+), mTEC (UEA1+),mTEClo (UEA-1+Ly51−), mTEChi(UEA-1+Ly51-MCHII+) is shown in both Mir181a1/b1fl/fl mice compared to Foxn1-Cre::Mir181a1/b1fl/fl. This experiment was done two times with at least 3 mice per experiment.
Fig 4.
Thymic profiles and numbers of cells are not altered due to loss of miR-181a1 and miR-181b1 in TECs.
Flow cytometric analysis for the cell-surface expression of CD4 and CD8 on thymocytes isolated from Mir181a1/b1fl/fl and Foxn1-Cre::Mir181a1/b1fl/fl mice. Numbers denote the percentage of cells within the given gates in a representative experiment. B. Thymocytes were harvested at two different time points 6 weeks (B) and 4 months (C). Absolute numbers are shown of the thymic subsets. There were no significant differences between Mir181a1/b1fl/fl (white) and Foxn1-Cre::Mir181a1/b1fl/fl mice (filled).
Fig 5.
Peripheral cell compartments are not affected by loss of miR-181a1 and miR-181b1 in TECs.
Spleen (A,C) and lymph nodes (B, D) were harvested at 6 weeks (A,B) and at 4 months (C,D). Absolute numbers are shown of the different subsets. There were no significant differences between Mir181a1/b1fl/fl (white) and Foxn1-Cre::Mir181a1/b1fl/fl mice (filled). This experiment was repeated twice with at least 5 mice in each group.