Fig 2: IFN-β facilitates TG02-induced inhibition of RNA polymerase II phosphorylation. Levels of pRNAPII, MCL-1, c-MYC and CDK9 were determined in LN-229 cells pretreated or not with IFN-β (1,000 IU/ml) for 24 h and then exposed to TG02 (100 nM) for 9, 24 or 72 h. β-actin served as loading control. pRNAPII, phosphorylated RNA polymerase II; ctr, control; IFN, interferon.

Fig 3: Inhibition of MYC sensitizes ALK+ NSCLC cells to crizotinibA. H2228, H3122 or STE-1 cells expressing either scrambled siRNA control (siSCR) or SmartPool siRNA targeting MYC (siMYC) were treated with crizotinib for 72 hours and proliferation was measured by MTS assay. B. Western blot demonstrates knockdown efficiency of MYC. C. Quantitative real-time PCR analysis of MYC in ALK+ cells after treatment with 5μM TMPyP4 for 48 hours. (**P < 0.01, *P < 0.05). D. Western blot analysis demonstrating c-MYC protein expression after treatment with 5μM TMPyP4 for up to 48 hours. E. ALK+ cells were treated with crizotinib in combination with either vehicle or 5μM TMPyP4 for 72 hours and proliferation was measured by MTS assay.

Fig 4: ALK regulates the MYC promoter and minimally regulates c-MYC protein stabilization in ALK+ NSCLC cellsA. Western blot analysis of c-MYC phosphorylation sites in ALK+ cell lines treated with 200nM TAE684 for up to 60 minutes. B. Western blot analysis of ALK+ cell lines treated with proteasome inhibitor MG132 alone or in combination with TAE684 for 0, 4, or 16 hours. C. Luciferase activity measuring MYC promoter activity in H3122 cells expressing HBM-Luc or SNM-Luc promoter construct treated with 200nM TAE684. (**P < 0.01). D. Luciferase activity measuring MYC promoter activity in NIH3T3 cells co-transfected with EML4-ALK or pCDH-Empty Vector and HBM-Luc constructs. Western blot showing expression of ALK in EML4-ALK-expressing NIH3T3 cells. (**P < 0.01). E. Luciferase activity measuring MYC promoter activity in NIH3T3 cells co-transfected with pCDH-empty vector or EML4-ALK wild-type (WT) or the cDNA encoding L1196M, G1269A or C1156Y mutations and the HBM-Luc MYC reporter construct. F. Western blot analysis of NIH3T3 lysates expressing the indicated cDNA vector treated with crizotinib.

Fig 5: Regulation of MYC and MYCBP expression is ALK-dependent in NSCLC cellsA. and B. Quantitative real-time PCR (qRT-PCR) analysis of MYC or MYCBP in NSCLC cells. NSCLC cell line panel includes ALK+ cell lines (H2228, H3122, STE-1), a ROS1 fusion line (HCC78), two KRAS cell lines (A549; G12S, H460; Q61H) and NRAS (H1299; codon 61), two mutant EGFR cell lines (H1650, HCC827; del 19). C. and D. Quantitative real-time PCR analysis of MYC or MYCBP in ALK+ cell lines in response to 24 hour treatment with 200nM TAE684. (**P < 0.01, *P < 0.05). E. Western blot analysis of H2228, H3122 and STE-1 cells after 24 hour treatment with 200nM TAE684. F. Western blot analysis of ALK+ cells treated for 24 hours with the indicated dose of crizotinib.