Figure 5.

Functional Validation of the Effects of Hopx on HSPC Physiology

(A) The frequency of LSKCD150⁻CD48⁻ cells, as a percentage of BM MNCs (left y axis), are significantly reduced in Hopx^−/− mice compared to wild-type littermate controls (Hopx^+/+), whereas LSKs are not affected (right y axis). BM MNCs were isolated from the femurs and tibias of 12-week-old male mice and the frequency of different HSPC sub-populations was determined by flow cytometry.

(B) Competitive repopulation assay shows significantly decreased levels of engraftment starting at 16 weeks after transplantation of HSCs isolated from BM of Hopx^−/− mice compared to wild-type Hopx^+/+ littermates. Mice were bled from the tail vein every 4 weeks after transplantation and the level of engraftment, indicated by the percentage of circulating CD45.2⁺ cells, was quantified by flow cytometry.

(C) Frequency of LSKCD150⁻CD48⁻ cells in BM, as a percentage of MNCs (left y axis), is significantly reduced in mice transplanted with HSCs from Hopx^−/− mice compared to Hopx^+/+ littermate controls 6 months following transplantation. There is no difference in the frequency of LSKs (right y axis).

(D) LSKCD150⁻CD48⁻ cells of Hopx^−/− mice exhibit altered cell-cycle status, with decreased and increased percentage of cells in the G₀ and G₁ phases, respectively, compared to wild-type littermates. No effects were observed in LSKs.

For each experiment in (A)–(D), error bars represent SEM and n = 3–5 mice per group. ^∗p < 0.05; ^∗∗p < 0.01. See also Figure S5.