Figure 4. Reducing Wnt5a expression in aged LT-HSCs rejuvenates their function in vivo.

a, Experimental set-up. b, B220⁺, CD3⁺ and myeloid cells among donor-derived cells in bone marrow. *P < 0.05; n = 10. c, LT-HSCs, ST-HSCs and LMPPs among donor-derived LSKs. *P < 0.05; **P < 0.01. d, e, Cdc42 activity in aged donor-derived low-density bone-marrow (LDBM) cells transduced with non-targeting (NT) shRNA, Wnt5a shRNA (Wnt5a^KD) and untransduced control (d) and densitometric score (e). n = 4, *P < 0.05. f, Cdc42 and tubulin in donor-derived LT-HSCs from aged untransduced or non-targeting shRNA or Wnt5a^KD recipient mice 24 weeks after transplant shown by immunofluorescence. Scale bar, 5 μm. g, Immunofluorescence z-stack and three-dimensional merged images of tubulin (green) and β-catenin (red) localization in aged non-targeting shRNA or Wnt5a^KD LT-HSCs. Scale bar, 5 μm. h, Aged donor-derived non-targeting shRNA or Wnt5a^KD LT-HSCs exhibiting nuclear accumulation of β-catenin. n = 3, *P < 0.05. A paired Student's t-test was used to determine the significance of the difference between means of two groups. One-way ANOVA or two-way ANOVA were used to compare means among three or more independent groups. Error bars represent s.e.m.