TLX1 and NOTCH coregulate MYC levels and ALL-SIL growth. (A) ALL-SIL derivatives expressing the indicated levels of TLX1 were treated for 24 hours with 500 nM Compound E (GSI, +) and 0.05% DMSO (GSI, -) as vehicle control. Whole cell lysates were analyzed for expression of activated NOTCH1 and MYC proteins by Western blotting. A representative blot of 4 biological replicates is shown. (B) Activated NOTCH1 expression does not compensate for the lack of TLX1. ALL-SIL cells expressing the TX1 shRNA95 or pLKO.1-CFP control vector were lentivirally transduced to express a constitutively active form of NOTCH1 (ICN1). Cells were seeded at 1 × 105 cells per ml in parallel with mock-transduced controls and counted on days 1 and 4 by trypan blue staining. (C) Growth competition experiments in the presence or absence of 500 nM Compound E, indicated as GSI and DMSO, respectively. ALL-SIL cells expressing TLX1 shRNA95 and CFP or pLKO.1-CFP were mixed with parental ALL-SIL cells in equal proportions. Mixed populations were divided, treated with GSI or DMSO control for up to 3 weeks and periodically examined by flow cytometric analysis. (D) TLX1 and NOTCH regulation of MYC and HES1 mRNA. (E) TLX1 prevents GSI-induced downregulation of MYC protein and extends MYC half-life. Cells were treated with 500 nM Compound E or DMSO control for 24 hours, then assayed for MYC protein levels and stability using 50 μg/ml cycloheximide or 40 μM MG132 treatment for the indicated times.