Acute systemic LPS treatment transiently increases HSC proliferation via TLR4. (A) Scheme indicating in vivo treatment of wt or TLR4^−/− mice with PBS (control) or LPS (0.25 mg/kg) for 18 hours. (B) Cell cycle analysis using intracellular (ic) Ki67-Hoechst 33342 staining (G₀ cells icKi67^neg Hoechst 33342^low; G₁ cells icKi67^pos Hoechst 33342^low; SG₂M cells icKi67^pos Hoechst 33342^hi) of HSCs (LSK CD150⁺CD48^-CD34^-) from wt and TLR4^−/− mice (n = 3) treated with PBS or LPS (0.25 mg/kg, 18 hours). P values refer to G₀ phase and were determined by ANOVA Tukey’s post hoc test. (C) Scheme indicating in vivo BrdU uptake (18 mg/kg, 14 hours) and treatment with PBS (control) or LPS (0.25 mg/kg) of wt or TLR4^−/− mice for 18 hours. (D) Fourteen-hour BrdU (18 mg/kg) uptake of HSCs (LSK CD150⁺CD48^-CD34^-) from PBS- (control) or LPS- (0.25 mg/kg, 18 hours) treated wt and TLR4^−/− mice (n = 6). P values were determined by ANOVA Tukey’s post hoc test. (E) Percentage of HSCs (LSK CD150⁺CD48^-CD34^-) in G₀ phase (icKi67^neg Hoechst 33342^low) at indicated time points after treatment of wt mice with PBS (control; time point 0 hours; blue dots) or LPS (0.25 mg/kg; green dots) (n = 6). P values were determined by ANOVA Tukey’s post hoc test. (F) Fourteen-hour BrdU (18 mg/kg) uptake of HSCs from wt (n = 12), Myd88^−/− (n = 6), or Trifmc^−/− (n = 5) mice treated with PBS (control) or LPS (0.25 mg/kg, 18 hours). P values were determined by unpaired t test. (G) Scheme illustrating the in vivo LRC assay: BrdU (18 mg/kg IP) uptake at day 1, subsequent 10-day BrdU pulse-period (1 mg/mL in drinking water), followed by a BrdU-free 12-week chase. Subsequently, mice were treated with PBS (control) or LPS (0.25 mg/kg) every third day. Analysis was done 2 weeks after last LPS injection. (H) Percentage of BrdU⁺ LRCs of LSK CD150⁺CD48^-CD34^- in wt and TLR4^−/− mice. Experimental set-up as indicated in Figure 1G (wt: n = 6 PBS; n = 10 LPS; TLR4^−/−: n = 4 PBS; n = 3 LPS). P values were determined by ANOVA Tukey’s post hoc test.