Ko indicates knock-out; I°, primary; II°, secondary; III°, tertiary; IV°, quaternary; ND, not done; and –, not applicable.

The cBMT procedure was detailed in our previous publication¹⁶ and is further illustrated in Figure 2A. Briefly, test cells (2 × 10⁶ BMNCs/host) from p18^–/– or p18^–/–p21^–/– male mice and an equal number of wild-type BMNCs (competitor cells) from male mice (wild-type litter mates in primary cBMT and then 2-month-old wild-type C57BL/6129/SvF1 mice in subsequent rounds of cBMT) were cotransplanted into lethally irradiated (10 Gy) female recipients. Overall engraftment levels in blood are presented in Figure 2C-D 

Absolute yield of KO CD34^–LSK cells was calculated according to the bone marrow cellularity per harvest (including ilia, femurs, and tibias; n = 8), the frequency (by flow cytometry) of viable CD34^–LKS (being able to respond to growth stimuli in vitro) and percentage of KO in CD34^–LKS cells. P < .01 (t test, between p18^–/– and p21^–/– cBMT groups). The lower yield of CD34^–LKS cells due to the absence of p21 must have been substantially underestimated since the p18^–/– group had undergone 2 additional rounds of cBMT

The representations of p18^–/– genotype after after secondary cBMT have been reported in our previous publication.¹⁶  All the mice after quaternary cBMT in the p18^–/– group developed T-cell leukemia. Characterization of the leukemic cells and relation to HSC regeneration involving a range of analyses are to be reported in a different manuscript⁴¹ 

This experiment was done much later than the other 2 experiments (p18^–/– and p18^–/–p21^–/–) to confirm the accelerated hematopoietic exhaustion due to p21 absence¹⁰  as rationalized in the legend of Figure 2D. The competitor cells in all these experiments were from the same type of unmanipulated bone marrow