Figure 5.
LPS-induced activation of quiescent HSCs depends on Sca-1. (A) Heatmap visualizing the ProcartaPlex Multiplex immunoassay on BM supernatant from wt mice treated with PBS (control) and LPS (0.25 mg/kg, 18 hours) (n = 4). (B) TNFα and IL-1β levels determined by ELISA in BM supernatants after in vivo treatment of wt mice with PBS (control) or LPS (0.25 mg/kg, 4 hours) (n = 3). P values were obtained by unpaired t test. (C) qRT-PCR analyzing cytokine gene expression in indicated cell populations from wt mice (n = 3) treated with PBS (control) or LPS (0.25 mg/kg, 4 hours). Populations are defined as high inflammatory monocytes (CD11b+Cd115+Ly6C+), resident monocytes (CD11b+CD115+Ly6C-), macrophages (Gr1-CD115intF4/80+), and neutrophils (CD11b+Gr1+CD115-). P values were obtained by unpaired t test. (D) Scheme indicating in vivo treatment of wt and Tnfrsf1−/− mice with PBS (control) or TNFα (0.75 mg/kg, 18 hours). (E) Cell cycle analysis (icKi67-Hoechst 33342) of HSCs from wt or Tnfrsf1−/− mice treated with PBS or TNFα (0.75 mg/kg, 18 hours) as indicated in Figure 5D (n = 6). P values refer to G0 phase and were determined by ANOVA Tukey’s post hoc test. (F) Scheme indicating in vivo treatment of wt and IL-1R−/− mice treated with PBS (control) or IL-1α or IL-1β (each 0.25 mg/kg) for 18 hours. (G) Cell cycle analysis (icKi67-Hoechst 33342) of HSCs (LSK CD150+CD48-CD34-) from wt or IL-1R−/− mice treated with PBS (control), IL-1α, or IL-1β (each 0.25 mg/kg, 18 hours) as indicated in Figure 5F (n = 3). P values refer to G0 phase and were determined by ANOVA Tukey’s post hoc test. (H) Scheme indicating treatment of wt and Sca-1−/− mice with PBS (control), TNFα (0.75 mg/kg), IL-1α, or IL-1β (each 0.25 mg/kg) for 18 hours. (I) Fourteen-hour BrdU (18 mg/kg) uptake of HSCs (LK CD150+CD48-CD34-) from wt or Sca-1−/− mice (n = 6) treated with PBS or TNFα (0.75 mg/kg, 18 hours). P values were determined by ANOVA Tukey’s post hoc test. (J) Cell cycle analysis (icKi67-Hoechst 33342) of HSCs (LK CD150+CD48-CD34-) from wt or Sca-1−/− mice treated with PBS, IL-1α, or IL-1β (each 0.25 mg/kg, 18 hours) (n = 3). P values refer to G0 phase and were determined by ANOVA Tukey’s post hoc test. (K) Sca-1 expression on HSPCs (LK CD150+) from wt mice treated with PBS, TNFα (0.75 mg/kg, 18 hours) (n = 6), IL-1α, or IL-1β (each 0.25 mg/kg, 18 hours) (n = 3). P values were determined by ANOVA Tukey’s post hoc test.

LPS-induced activation of quiescent HSCs depends on Sca-1. (A) Heatmap visualizing the ProcartaPlex Multiplex immunoassay on BM supernatant from wt mice treated with PBS (control) and LPS (0.25 mg/kg, 18 hours) (n = 4). (B) TNFα and IL-1β levels determined by ELISA in BM supernatants after in vivo treatment of wt mice with PBS (control) or LPS (0.25 mg/kg, 4 hours) (n = 3). P values were obtained by unpaired t test. (C) qRT-PCR analyzing cytokine gene expression in indicated cell populations from wt mice (n = 3) treated with PBS (control) or LPS (0.25 mg/kg, 4 hours). Populations are defined as high inflammatory monocytes (CD11b+Cd115+Ly6C+), resident monocytes (CD11b+CD115+Ly6C-), macrophages (Gr1-CD115intF4/80+), and neutrophils (CD11b+Gr1+CD115-). P values were obtained by unpaired t test. (D) Scheme indicating in vivo treatment of wt and Tnfrsf1−/− mice with PBS (control) or TNFα (0.75 mg/kg, 18 hours). (E) Cell cycle analysis (icKi67-Hoechst 33342) of HSCs from wt or Tnfrsf1−/− mice treated with PBS or TNFα (0.75 mg/kg, 18 hours) as indicated in Figure 5D (n = 6). P values refer to G0 phase and were determined by ANOVA Tukey’s post hoc test. (F) Scheme indicating in vivo treatment of wt and IL-1R−/− mice treated with PBS (control) or IL-1α or IL-1β (each 0.25 mg/kg) for 18 hours. (G) Cell cycle analysis (icKi67-Hoechst 33342) of HSCs (LSK CD150+CD48-CD34-) from wt or IL-1R−/− mice treated with PBS (control), IL-1α, or IL-1β (each 0.25 mg/kg, 18 hours) as indicated in Figure 5F (n = 3). P values refer to G0 phase and were determined by ANOVA Tukey’s post hoc test. (H) Scheme indicating treatment of wt and Sca-1−/− mice with PBS (control), TNFα (0.75 mg/kg), IL-1α, or IL-1β (each 0.25 mg/kg) for 18 hours. (I) Fourteen-hour BrdU (18 mg/kg) uptake of HSCs (LK CD150+CD48-CD34-) from wt or Sca-1−/− mice (n = 6) treated with PBS or TNFα (0.75 mg/kg, 18 hours). P values were determined by ANOVA Tukey’s post hoc test. (J) Cell cycle analysis (icKi67-Hoechst 33342) of HSCs (LK CD150+CD48-CD34-) from wt or Sca-1−/− mice treated with PBS, IL-1α, or IL-1β (each 0.25 mg/kg, 18 hours) (n = 3). P values refer to G0 phase and were determined by ANOVA Tukey’s post hoc test. (K) Sca-1 expression on HSPCs (LK CD150+) from wt mice treated with PBS, TNFα (0.75 mg/kg, 18 hours) (n = 6), IL-1α, or IL-1β (each 0.25 mg/kg, 18 hours) (n = 3). P values were determined by ANOVA Tukey’s post hoc test.

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