HSC activation is mediated by cell-extrinsic TLR4 signaling. (A) Scheme indicating in vivo transplantation: BM from wt (CD45.1/2⁺) and TLR4^−/− mice (CD45.2⁺) (1.5 × 10⁶ BM cells each) was transplanted into irradiated wt mice (CD45.1⁺). Mice were treated with PBS (control) or LPS (0.25 mg/kg, 18 hours) 12 weeks after transplantation. (B) Cell cycle analysis (icKi67-Hoechst 33342) of wt and TLR4^−/− HSCs (LSK CD150⁺CD48^-CD34^-) in mixed BM chimeras after PBS (control) or LPS treatment as indicated in Figure 2A (n = 3). P values refer to G₀ phase and were determined by ANOVA Tukey’s post hoc test. (C) Relative Sca-1 expression in LK CD150⁺ cells (HSPCs) of wt and TLR4^−/− mice treated with PBS (control) or LPS (0.25 mg/kg, 18 hours) (n = 8) and representative FACS profile. P values were determined by ANOVA Tukey’s post hoc test. (D) Scheme indicating in vitro treatment or sorted HSPCs (LK CD150⁺) with PBS (control), IFN-α (1000 U/mL), or LPS (100 ng/mL). (E) Relative Sca-1 expression on sorted HSPCs (LK CD150⁺) after PBS (control), IFN-α, or LPS in vitro treatment for 18 hours as indicated in Figure 2D (n = 3). P values were determined by ANOVA Tukey’s post hoc test.