Figure 2. HSCs directly respond to LPS in vitro and in vivo.

(A) TLR4 expression on CD150⁺CD48⁻ LSK LT-HSCs by flow cytometry. Bone marrow-derived macrophages (BMDM) are a positive control. Open histogram, isotype control; shaded histogram, TLR4 staining. Data are representative of 3 independent experiments. (B) Sort purified LT-HSCs from WT or TLR4 KO mice were pulsed with 10 μg/ul LPS for 12 hours in serum free medium and then stained with antibodies to BrdU. n=3 independent cell sorts. (C) Mixed WT:TLR4^KO BM chimeras were exposed to LPS or vehicle every other for 4–6 weeks after which BM LT-HSCs and CLPs were enumerated. Two separate adoptive transfers were performed in which competitive donors were engrafted into six total mice in each experiment. Each donor is a different symbol. CLPs are depicted for all mice; one set of HSC samples was lost due to flow cytometry clogging. Data (avg ± SEM) were analyzed by paired t-test. **p<0.05; ns, not significant.