Role of MYC as a downstream component of the TLX1/NOTCH regulatory network. (A) Chemical inhibition of MYC (10058-F4 treatment) mimics TLX1 knockdown in ALL-SIL cells expressing shRNA93 versus pLKO.1-CFP expressing TLX1+ controls. (B) Expression of wild-type but not an inactive MYC mutant (MYC ΔC) compensates for the lack of TLX1 expression in ALL-SIL cells treated with GSI. TLX1 shRNA93 was co-expressed with CFP; MYC constructs were coexpressed with RFP. ALL-SIL cells expressing pLKO.1-CFP-TLX1 shRNA93 were transduced with retroviral vectors coexpressing RFP alone, wild-type MYC plus RFP or the MYC ΔC mutant plus RFP and sorted for CFP and RFP expression. Equal numbers were seeded and counted after 2 weeks in culture. CFP + RFP, parental ALL-SIL cells coexpressing CFP and RFP. (C) Growth competition experiments. The same CFP+RFP+ ALL-SIL derivatives as in B were mixed with parental ALL-SIL cells expressing GFP in equal proportions. Aliquots were periodically analyzed by flow cytometry; shown are data 2 weeks after initiation of the experiment. (D) Western blot analysis showing MYC protein levels and the corresponding levels of TLX1 for the cell lines studied in B and C.