Figure 7

Tlx1 is required for LPS-induced EMH in the spleen. (a) Schematic of the experimental strategy including time points of the analysis. Tlx1^{CreER-Venus/+} littermate control mice (Ctr) and Tlx1^{CreER-Venus/fl} mice (CKO) treated with tamoxifen for 5 consecutive days were administered LPS or endotoxin-free saline for an additional 3 consecutive days and then analyzed 48 hours after the final treatment. Data were pooled from at least 2 independent experiments. (b) Gross appearance (left), weight (middle) and total cell number (right) of the spleen from Ctr and CKO mice treated with LPS or saline. (n = 3–4). (c) Total cell numbers of the indicated hematopoietic cell populations from the spleen of Ctr and CKO mice treated with saline or LPS. The gating strategy was same as in Fig. 3a. (n = 3–4). (d) Total cell numbers of the indicated hematopoietic stem/ progenitor cell populations in the BM of Ctr and CKO mice treated with saline or LPS. The gating strategy was same as in Fig. 2f,g. (n = 3–4). (e) The percentage of the indicated hematopoietic stem/ progenitor cell populations in the peripheral blood of Ctr and CKO mice treated with saline or LPS. (n = 3). (f) Serum G-CSF concentration in Ctr and CKO mice treated with saline or LPS. (n = 3). (g) Representative flow cytometric profiles of CD45⁻ Ter119⁻ CD31⁻ stromal cells from the spleen of Ctr and CKO mice treated with saline or LPS. Gates used to identify Venus⁺ cell populations are outlined and numbers above the outlined areas indicate percent events in each gate. The histogram represents the intensity of fluorescence of Venus⁺ cells from Ctr mice treated with LPS (red line) or saline (black line), and CKO mice treated with LPS (blue). (h) The MFI and total cell numbers of Venus⁺ cells of Ctr and CKO mice treated with saline or LPS. The relative MFI of Venus⁺ cells in the saline-treated Ctr was arbitrarily set to 1. (n = 3–6). (i) Expression of CXCL12 and SCF mRNA in Venus⁺ and Venus⁻ cells among CD45⁻ Ter119⁻ CD31⁻ splenic stroma cells from Ctr and CKO mice 24 hours after tamoxifen treatment. Data were normalized to β-actin and the level of mRNA transcripts in Venus⁺ cells of Ctr mice was arbitrarily set to 1. (n = 3). (j) Expression of CXCL12 and SCF mRNA in splenic Venus⁺ and Venus⁻ cells of saline-treated Ctr mice, LPS-treated Ctr and LPS-treated CKO mice. Data were normalized to β-actin and the level of mRNA transcripts in Venus⁻ cells of saline-treated Ctr mice was arbitrarily set to 1. (n = 3). (k) Clustering analysis of Venus⁺ cells in the spleen from Ctr and CKO mice treated with LPS or saline. Representative digital images of Venus⁺ cells (white dots) in the spleen sections from the indicated mice. The right graph represents the Hopkins index of Venus⁺ cells in the spleen of Ctr and CKO mice treated with saline or LPS. (n = 3).