Figure 2.
USP15 Depletion Impairs HSPC Proliferation In Vitro and Reconstitution Potential In Vivo
(A) In vitro and in vivo validation assays for USP15-targeting shRNAs.
(B) Knockdown efficiency of shRNAs targeting USP15 in Lin− cells as measured by qRT-PCR. Mean values of three technical replicates ± SD are shown.
(C) Flow cytometry analysis of Lin− cells at 1 week post-infection. The frequency of LSKs in the Lin−, c-Kit+ population, as well as the frequency of Lin−, c-Kit+, and LSKs in the live culture, was calculated and normalized relative to shScramble control. n = 3 independent experiments. Mean values ± SEM are shown.
(D) Freshly purified Lin− cells were plated 7 days post infection and monitored for growth. n = 4 wells per data point. Mean values ± SEM are shown.
(E–K). Freshly isolated WT Lin− cells transduced with the indicated shRNAs were assayed in competitive BM transplantation. Mean values ± SEM are shown. n = 3 per shRNA, except for shUSP15#16 (n = 4).
(E and F) CD45.2 chimerism in peripheral blood (E) and contribution of transduced cells to myeloid (Gr1+), B cell (CD19+), and T cell (CD3+) lineages in the blood (F) of recipients. PBC, peripheral blood cells.
(G) CD45.2 chimerism in B cell and T cell lineages in recipients’ spleen at 18 wpt.
(H) Representative FACS profiles of the LSK compartment in recipients at 18 wpt.
(I) CD45.2 chimerism level in LSKs in primary recipients (left). Right: numbers of donor-derived LSKs in 106 viable BM cells at 18 wpt.
(J) Cell numbers of donor-derived HSCs (LSK/CD150+/CD48−) in 106 viable BM cells at 18 wpt.
(K) Fraction of donor-derived LKSs−, CMPs, GMPs, and MEPs in primary recipients at 18 wpt.
∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001. p value was assessed by Student’s t test or multiple t test (D) in Prism 7. See also Figure S3.