Figure 4.
Acute LPS exposure depends on IFN signaling to activate HSCs. (A) Scheme illustrating in vitro culturing of HSPC (LK CD150+) and CD11b+ CD115+ BM cells in presence of LPS (100 ng/mL, 18 hours) and subsequent transfer of conditioned medium to cultured wt or TLR4−/− HSPCs (18 hours). (B) Relative Sca-1 expression in LK CD150+ cells (HSPCs) after in vitro culturing with conditioned medium as indicated in Figure 4A (n = 3). P values were obtained by unpaired t test. (C) Scheme indicating in vivo BrdU uptake (18 mg/kg, 14 hours) and treatment of wt and Sca-1−/− mice with PBS (control) or LPS (0.25 mg/kg) for 18 hours. (D) Fourteen-hour BrdU (18 mg/kg) uptake of HSCs (LK CD150+CD48-CD34-) from wt or Sca-1−/− mice treated with PBS or LPS (0.25 mg/kg) for 18 hours (n = 6). P values were obtained by unpaired t test. (E) IFNα and IFNγ levels determined by ELISA in BM supernatant after in vivo treatment of wt mice with PBS or LPS (0.25 mg/kg, 4 hours) (n = 6). P values were determined by unpaired t test. (F) Principal component analysis and comparison of Illumina Chip array data obtained from HSCs isolated from PBS (control), LPS (0.25 mg/kg), or IFNα (5 × 106U/kg) treated wt mice (18 hours). (G) Hallmark pathway enrichment analysis using EnrichR of 88 differentially expressed genes (DEGs) shared between IFNα– and LPS-treated HSCs. P values were obtained from the online EnrichR program and were not modified. (H) Gene ontology biological processes enrichment analysis using EnrichR of 88 DEGs shared between IFNα– and LPS-treated HSCs. P values were obtained from the online EnrichR program and were not modified. (I) Scheme indicating in vivo treatment of wt and IFNAR−/−IFNGR−/− mice with PBS (control) or LPS (0.25 mg/kg) for 18 hours. (J) Cell cycle analysis (icKi67-Hoechst 33342) of HSCs (LSK CD150+CD48-CD34-) from wt or IFNAR−/−IFNGR−/− mice treated with PBS (control) or LPS (indicated concentrations, 18 hours). Experimental set-up as indicated in Figure 4I (n = 3). P values refer to G0 phase and were determined by ANOVA Tukey’s post hoc test.

Acute LPS exposure depends on IFN signaling to activate HSCs. (A) Scheme illustrating in vitro culturing of HSPC (LK CD150+) and CD11b+ CD115+ BM cells in presence of LPS (100 ng/mL, 18 hours) and subsequent transfer of conditioned medium to cultured wt or TLR4−/− HSPCs (18 hours). (B) Relative Sca-1 expression in LK CD150+ cells (HSPCs) after in vitro culturing with conditioned medium as indicated in Figure 4A (n = 3). P values were obtained by unpaired t test. (C) Scheme indicating in vivo BrdU uptake (18 mg/kg, 14 hours) and treatment of wt and Sca-1−/− mice with PBS (control) or LPS (0.25 mg/kg) for 18 hours. (D) Fourteen-hour BrdU (18 mg/kg) uptake of HSCs (LK CD150+CD48-CD34-) from wt or Sca-1−/− mice treated with PBS or LPS (0.25 mg/kg) for 18 hours (n = 6). P values were obtained by unpaired t test. (E) IFNα and IFNγ levels determined by ELISA in BM supernatant after in vivo treatment of wt mice with PBS or LPS (0.25 mg/kg, 4 hours) (n = 6). P values were determined by unpaired t test. (F) Principal component analysis and comparison of Illumina Chip array data obtained from HSCs isolated from PBS (control), LPS (0.25 mg/kg), or IFNα (5 × 106U/kg) treated wt mice (18 hours). (G) Hallmark pathway enrichment analysis using EnrichR of 88 differentially expressed genes (DEGs) shared between IFNα– and LPS-treated HSCs. P values were obtained from the online EnrichR program and were not modified. (H) Gene ontology biological processes enrichment analysis using EnrichR of 88 DEGs shared between IFNα– and LPS-treated HSCs. P values were obtained from the online EnrichR program and were not modified. (I) Scheme indicating in vivo treatment of wt and IFNAR−/−IFNGR−/− mice with PBS (control) or LPS (0.25 mg/kg) for 18 hours. (J) Cell cycle analysis (icKi67-Hoechst 33342) of HSCs (LSK CD150+CD48-CD34-) from wt or IFNAR−/−IFNGR−/− mice treated with PBS (control) or LPS (indicated concentrations, 18 hours). Experimental set-up as indicated in Figure 4I (n = 3). P values refer to G0 phase and were determined by ANOVA Tukey’s post hoc test.

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