Aged hematopoietic stem cells exhibit compromised mobility of FUS. (A) Representative western blot showing the expression of FUS in young and aged HSCs. Freshly isolated 20 000 CD34^– LSK (Lineage⁻ Sca-1⁺ c-Kit⁺, hematopoietic stem and progenitor cell-enriched population) cells from young (2 months) and aged (24-28 months) wild-type (WT) mice were lysed in 2 × sodium dodecyl sulfate loading buffer. Lysis was completed by sonication, denatured by boiling, and subjected to a western blot assay to detect FUS. (B) Representative western blot showing the expression of FUS in FUS-overexpressed LSK cells. Freshly isolated LSK cells from young (2 months) WT mice were infected by FUS-carrying lentivirus (Fus OE) and empty vector (EV), and 3 days later, 10⁵ GFP⁺ cells were purified for western blot assay with indicated antibodies. (C) Experimental design of a competitive transplantation assay for FUS-overexpressed HSCs. Freshly isolated 10⁵ LSK cells from young WT mice (2 months) were infected by FUS-carrying lentivirus (Fus OE). Three days later, 2000 GFP⁺ Sca-1⁺CD48^– cells were purified and transplanted into lethally irradiated recipients (CD45.2) together with 3.5 × 10⁵ competitor cells (CD45.1). Secondary transplantation was performed by using 2 × 10⁶ total BM cells from the primary recipients. Chimera in peripheral blood was evaluated every 4 weeks until the 16th week. (D) These line plots depict the percentage of donor-derived cells (overall, B cell, T cell, myeloid cell) in peripheral blood of primary recipients at indicated time points. Refer to supplemental Figure 1E. N = 8 recipients per group, data are shown as mean ± SEM. (E) The histogram depicts the lineage distribution of donor-derived peripheral blood cells at the 16th week. N = 8 recipients per group, data are shown as mean ± SEM. Refer to supplemental Figure 1F. (F) Experimental design of competitive transplantation assay. Freshly isolated 10⁵ LSK cells from young (2 months) and aged (18 months) WT mice were infected by shFus-carrying lentivirus, which is labeled by mCherry fluorescence. Three days later, 2000 mCherry⁺Sca-1⁺CD48^– cells were purified and transplanted into lethally irradiated recipients (CD45.2) together with 3.5 × 10⁵ competitor cells (CD45.1). Chimera in peripheral blood was evaluated every 4 weeks until the 16th week. (G) Representative western blot showing the knockdown efficiency of shRNA against Fus. Freshly isolated 2 × 10⁴ HSCs (mCherry⁺ CD48^– LSK) from the recipients of panel F at the end of the 16th week after transplantation (left) and 2 × 10⁴ cultured mCherry⁺ LSK from young WT mice (right) were lysed for western blot assay with indicated antibodies. (H) These line plots depict the percentage of donor-derived cells (overall, B cell, T cell, myeloid cell) in peripheral blood of primary recipients at indicated timepoints. N = 7 recipients per group, data are shown as mean ± SEM. (I) The histogram depicts the lineage distribution of donor–derived peripheral blood cells at the 16th week. N = 7 recipients per group, data are shown as mean ± SEM. (J) Representative fluorescence recovery after photobleaching (FRAP) images show the fluorescence recovery of FUS condensates in cytoplasm of HSCs (CD34^–LSK) from young (2 months) and aged (18 months) Fus-gfp mice after 0.5 mM ARS treatment for 6 hours. Scale bar represents 2 μm. Refer to supplemental Video 1. (K) The line plot depicts the recovery rate of FUS condensates in young (N = 21) and aged (N = 26) HSCs of panel J at indicated time points. Data are shown as mean ± SEM. (L) The histograms depict the mobile fraction (71.5% vs 28.7%) and T-half (4.3% vs 54.4%) of young and aged HSCs. T-half is the half-maximal recovery time. Data are shown as mean ± SEM. (M) Representative FRAP images show the fluorescence recovery of FUS condensates in the cytoplasm of shCtl-young, shCtl-aged, and shFUS-aged LSK cells after 0.5 mM ARS treatment for 6 hours. (N) The line plot depicts the recovery rate of FUS condensates in shCtl-young (N = 21), shCtl-aged (N = 15), and shFUS-aged (N = 13) LSK cells of panel M at indicated time points. (O) The histograms depict mobile fraction and T-half of shCtl-young, shCtl-aged, and shFUS-aged LSK cells. Data are shown as mean ± SEM. SEM, standard error of the mean. Freshly isolated 2 × 10⁴ HSCs (mCherry⁺ CD48^– LSK) from the recipients of panel F at the end of the 16th week after transplantation (left) and 2 × 10⁴ cultured mCherry⁺ LSK from young WT mice (right) were lysed for western blot assay with indicated antibodies. Freshly isolated 2 × 10⁴ HSCs (mCherry⁺ CD48^– LSK) from the recipients of panel F at the end of the 16th week after transplantation (left) and 2 × 10⁴ cultured mCherry⁺ LSK from young WT mice (right) were lysed for western blot assay with indicated antibodies. GFP, green fluorescent protein.