Ncam1 expression confers stress resistance and prevents LSC exhaustion. (A-B) Clonogenic assays using isogenic B6-M/A9 and N^−/−-M/A9 cells. Replating was performed every 7 days (A) and every 10 days (B). The number of colonies was compared by 2-tailed Student t test. Error bars represent mean of 3 to 5 biological replicates ± SD of mean. (C) Kaplan-Meier survival curves of secondary transplanted animals (100 000 cells per mouse). Survival of B6-M/A9 (n = 10) or N^−/−-M/A9 (n = 11) recipients was compared by the log-rank test. (D) Left, Representative examples of spleens derived from leukemic mice. Right, Statistics of spleen sizes derived from secondary transplanted B6-M/A9 or N^−/−-M/A9 mice. (E) Left, representative scatter plots of CD117 expression; right, statistics showing the percentage of Lin⁻Kit^high B6-M/A9 (N = 8) or N^−/−-M/A9 (N = 8) AML cells after secondary transplantation. (F) Limiting-dilution transplantation experiment. Animals were transplanted with different numbers of BM cells derived from secondary recipients as indicated (1 × 10⁵: n = 3; all other groups: n = 5). Red/purple colors represent wild-type animals, blueish colors Ncam1-depleted animals. Left, Survival of each group is shown as Kaplan-Meier curve. The estimated LSC number was calculated by L-Calc (see “Materials and methods”). Left, Corresponding extreme limiting dilution analysis graph.