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. Author manuscript; available in PMC: 2016 Mar 3.
Published in final edited form as: Leukemia. 2015 Oct 9;30(3):728–739. doi: 10.1038/leu.2015.275

Figure 7. UBASH3B/Sts-1 promotes the growth of AML1-ETO cells through the interaction with CBL.

Figure 7

A. Two shRNAs showed efficient UBASH3B/Sts-1 knockdown and growth-inhibitory effect in AML1-ETO cells. AML1-ETO cells were transduced with NT or shUBASH and were cultured with cytokines. Changes in frequency of Venus+ cells (shRNA-transduced cells) in two independent AML1-ETO cell cultures are shown. Results are normalized to the frequency of Venus+ cells at day 3, set to 1. B. (Left) Interaction between endogenous CBL and UBASH3B/Sts-1 in AML1-ETO cells irrespective of cytokine stimulation. NT- or shCBL-transduced AML1-ETO-expressing CB cells were left unstimulated or stimulated with 6 cytokines (SCF, TPO, FLT3L, IL-3, IL-6, GM-CSF) for 5 minutes. Total cell lysates were immunoprecipitated with anti-CBL antibody, and CBL-bound UBASH3B/Sts-1 was detected by western blotting. (Right) NT- or shUBASH-1-transduced AML1-ETO-expressing CB cells were left unstimulated or stimulated with 6 cytokines for 5 minutes. Total cell lysates were immunoprecipitated with anti-phospho-tyrosine antibody, and tyrosine phosphorylated CBL was detected by western blotting. s.e., short exposure; l.e., long exposure. C. AML1-ETO cells were transduced with shUBASH-1 or shUBASH-2 (marked with Venus) in combination with vector or CBL mutants (Y371S or ΔE8/9, marked with GFP), and the growths of Venus+ (shRNA only) and GFP+Venus+ [shRNA + (vector or Y371S or ΔE8/9)] cells were monitored. Results are normalized to the frequency of Venus+ cells or that of GFP+Venus+ cells at day 2, set to 1. Y371S and ΔE8/9 fully reversed the negative effect of shUBASH-1, and partially reversed that of shUBASH-2. See also Figure S13C.