Fig. 3

HCK is required for leukaemogenesis and self-renewal of LSCs. (a-c) Genome PCR, RT-PCR, and western blotting were performed with HCK ^−/− BM cells to confirm HCK deletion. NC: Negative control; PC: Positive control.  (d) Experimental scheme for investigating the function of HCK in leukaemogenesis. (e) Frequency of YFP⁺ leukaemia cells in the peripheral blood at 32 days after the second transplantation. (f) Representative images of the sizes of spleens of recipient mice upon the second transplantation. (g) Quantification of the weights of the spleens in f. (h) Histological H&E staining of the spleens in f. (i) Representative images of colonies formed by YFP⁺c-Kit⁺ LSCs from secondary recipients. (j) The colony numbers were calculated in i. (k) Survival data for recipient mice receiving HCK^−/− or HCK^+/+ YFP⁺c-Kit⁺ LSCs upon the second transplantation (n = 10; log-rank test). (l) Frequency of YFP⁺ leukaemia cells in the peripheral blood at 32 days after the third transplantation. (m) Survival data for recipient mice receiving HCK^−/− and HCK^+/+ YFP⁺c-Kit⁺ LSCs upon the third transplantation (n = 5; log-rank test). (n) Representative images of colonies formed by YFP⁺c-Kit⁺ LSCs from the third transplant recipient. (o) The colony numbers were calculated in n. (p) Representative FACS plot and graph of c-Kit and Gr1 expression in HCK^−/− and HCK^+/+ leukaemic cells to assess the frequency of LSC. (q) Cell cycle status was determined in the third transplant recipient in vivo. (r) LSC frequency was determined from a limiting dilution assay performed with BM cells from the third transplant recipient mice. The ELDA web tool was used to calculate the frequency of LSCs