Fig 1: Infection with S. typhimurium drives long-chain fatty-acid uptake in HSC.a Schematic diagram of experimental design. WT CD45.2 lineage negative, CD117-positive (LK) cells were isolated and transduced with firefly luciferase (LK+FF) and transplanted into WT CD45.1 animals. b Mice were imaged using bioluminescence imaging to confirm engraftment. c Mice were injected with control PBS for 16 hours then treated with FFA-SS-luc and imaged using bioluminescence (FFA-luciferin). One-week later mice were injected LPS for 16 hours then treated with FFA-SS-luc and imaged using bioluminescence Representative images of control and LPS-treated mice. d Densitometry of the bioluminescent images in (c) to determine fluorescence intensity in the vehicle and LPS-treated animals. n = 4. e Schematic diagram of experiment in which C57BL/6 J mice were infected with S. typhimurium (Sal) for 72 hours and analyzed for LSK, MPP, HSC, ST-HSC, and LT-HSC populations by flow cytometry. f The gating strategy used to identify the LSK, MPP, HSC ST-HSC, LT-HSC populations are shown. g Lipid content (Bodipy 493/503 mean fluorescence intensity (MFI)) was assessed by flow cytometry from control and S. typhimurium (Sal) (72 hours) treated mice. n = 7 in each group. h C57BL/6 J mice were treated with 1 mg/kg LPS for 16 hours, the bone marrow was extracted, and the cells were analyzed by flow cytometry for lipid content (Bodipy 493/503 MFI) n = 6 in each group. i C57BL/6 J mice were infected with S. typhimurium (Sal) for 72 hours or LPS for 16 hours. Long-chain fatty-acid (LCFA) uptake was measured using the QBT assay. n = 5 in each group (j) C57BL/6 J mice were infected with S. typhimurium (Sal) for 72 hours. Representative live-cell fluorescent microscopy images of LSK cells isolated from the mice, Sca1 membrane stain (red), Bodipy 493/503 (green), and Hoechst 33342 (blue). Quantification of Bodipy 493/503 fluorescence in LSK cells from images shown, 20 LSK cells from five mice in each condition. Data shown are means ± SD. The Mann–Whitney U test (two-tailed) was used to compare between treatment groups *p < 0.05 **p < 0.01 ***p < 0.001. Source data are provided as a Source Data file.
Fig 2: FFA uptake through CD36 is an essential component of HSC expansion in response to infection.a Schematic diagram of experimental design. CD36+/+ CD45.1 lineage negative, CD117-positive cells were isolated and transplanted into CD36−/− CD45.2 animals. Post engraftment mice were treated with 1 mg/kg LPS for 16 hours and the bone marrow cells were analyzed by flow cytometry. b Flow cytometry analysis of CD36 expression (CD36 mean fluorescence intensity (MFI)) in the HSC from the transplant mice following 1 mg/kg LPS (16 hours) treatment. n = 5 mice in each group. c The LK cells were isolated and long-chain fatty-acid (LCFA) uptake was measured using the QBT assay. n = 5 in each group. d Percentage of cycling HSCs as measured by Ki67-positive cells from transplant mice after 16 hours of 1 mg/kg LPS treatment. n = 5 mice in each group. e The LSK population was isolated by FACS, oxygen consumption rate (OCR) was measured by the extracellular flux assay. Basal extracellular acidification rate (ECAR) compared with basal OCR levels provides a snapshot of the bioenergetic profile of LSK before and after treatment with LPS (16 hours). Basal OCR normalized to rotenone. n = 5 mice in each group. f Basal (normalized to rotenone) and maximal mitochondrial respiration in LSK cells from control vs. LPS 16 hours treated transplanted mice. n = 5 mice in each group. g CD36+/+ CD45.2 or CD36−/− CD45.2 lineage negative, CD117-positive cells were isolated and transplanted into WT CD45.1 mice these were termed WT(+/+CD36) or WT(−/−CD36). Post engraftment mice were treated with S. typhimurium for 96 hours. h Kaplan–Meier survival curve n > 5 mice in each group. i Weight loss was analyzed. n = 5 in each group. j Levels of circulating alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum following 96 hours of S. typhimurium treatment. n > 5 in each group. k Livers were isolated and sectioned and stained with hematoxylin and eosin. (Magnification: H, ×63). n = 5 mice in each group. Data shown are means ± SD. The Mann–Whitney U test (two-tailed) was used to compare between treatment groups *p < 0.05 **p < 0.01. Source data are provided as a Source Data file.
Fig 3: Infection increases dependency on ß-oxidation.a Schematic diagram of experimental design in which C57BL/6 J mice were infected with S. typhimurium (Sal) for 72 hours and 10 mg/kg/day Etomoxir (Eto) or 1 mg/kg LPS for 16 hours and 10 mg/kg Eto. The bone marrow was extracted, and the cells were analyzed by flow cytometry for LSK and HSC. b Percentage of cycling HSC and LSK as measured by Ki67-positive cells after 72 hours of S. typhimurium and 10 mg/kg/day Eto treatment. n > 5 mice in each group. c Percentage of cycling HSC and LSK as measured by Ki67-positive cells after 16 hours of 1 mg/kg LPS and 10 mg/kg Eto treatment. n > 4 mice in each group. d Number of LSKs and HSCs leg after 72 hours S. typhimurium and Eto treatment. n > 5 in each group. e Number of LSKs and HSCs per leg after 16 hours of LPS and Eto treatment. n = 5 mice in each group. f C57BL/6 J mice were treated with 1 mg/kg LPS for 16 hours or S. typhimurium for 72 h, HSC were FACS-sorted from control and LPS-treated animals. RNA was analyzed for CPT1A gene expression by qPCR. n = 4 in each group. g Schematic diagram of experimental design. WT CD45.1 lineage negative, CD117-positive (LK) cells were transduced with a CPT1A knockdown lentivirus (LKCPT1A KD) were transplanted into WT CD45.2 animals. Post engraftment mice were treated with 1 mg/kg LPS for 16 hours. h The bone marrow was extracted, and analyzed by flow cytometry for the LSK and HSC population. i Percentage of cycling HSC and LSK as measured by Ki67-positive cells after 16 hours of 1 mg/kg LPS treatment. n = 5 mice in each group. j Number of LSKs and HSCs per leg after 16 hours of LPS treatment. n = 5 mice in each group. Data shown are means ± SD. The Mann–Whitney U test (two-tailed) was used to compare between two treatment groups and the Kruskal–Wallis test was followed by Dunn’s multiple comparison post hoc test to compare between three treatment groups. *P < 0.05 **p < 0. 01. Source data are provided as a Source Data file.
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