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Table S1. List of genes targeted by the sequence-verified shRNAs identified in two independent cell-based systems. For candidate validation, multiple RNAi agents directed against independent sequences within each gene target were tested for modification of FOXO-driven luciferase activity. Gene targeting region for the silencing pools are shown. ORF, open reading frame; 5'UTR and 3'UTR, five-prime and three-prime untranslated regions, respectively.

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Table S2 RNAi agents used for the validation of TRIB2 silencing. In order to rule out off-target effects several independent TRIB2 interfering agents from different sources were tested and their knockdown efficiency against TRIB2 mRNA was analyzed by qRT-PCR. 1- Both shRNAs obtained from the NKI library initially included in the same pool. For further characterization both oligos were isolated and characterized independently. 2- This pool contains four different siRNAs. ORF, open reading frame; 5'UTR, five-prime untranslated regions.

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Table S3 Description of samples included in the cancer profiling array. Information about the cDNAs and controls immobilized on the cancer profiling array and the corresponding hybridization signals specific for TRIB2. Normalization was performed against signals obtained using the ubiquitin control probe.

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Table S4 Description of samples included in the Tissue qPCR Arrays. qRT-PCR was performed using TissueScan Melanoma Tissue qPCR Array I containing 43 tissues (OriGene Technologies, Rockville, MD). Quantification of TRIB2 mRNA levels was performed using qPCR. qRT-PCR amplification signals specific for TRIB2 were normalized against GAPDH expression.

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Fig. S1. A. General structure of the construct used to generate the 293foxREP cells. B. Validation of the 293foxREP system. FOXO-driven expression of luciferase was determined upon activation or inhibition of the PI3K/Akt pathway. Each construct was transiently co-transfected with plasmids encoding FOXO3a or constitutively active FOXO3a-(A)3 mutant and Renilla luciferase into 293foxREP cells, and the luciferase activities were measured as described above. The data were normalized to the Renilla luciferase (phRG-TK vector) reporter construct. The results are given as the mean ± SEM of three independent experiments performed in triplicate.

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Fig. S2. Schematics of the strategy used to screen the NKI library of shRNAs for novel modulators of FOXO activity. The shRNA library was introduced in pools of three RNAi per gene into 293foxREP cells. Those pools of shRNAs that caused activation of FOXO driven luciferase activity were analyzed for its capacity to induce nuclear translocation of FOXO in the U2foxRELOC system.

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Fig. S3. A and B. The Cancer Profiling Array II membrane after hybridization with the TRIB2 and ubiquitin probes. Tissue sources are listed above each sample group. C. Relative abundance of TRIB2 as compared with GAPDH expression as determined by comparison of TRIB2 and GAPDH qRT-PCR amplification signals. cDNAs studied were normal skin tissues (n=3), malignant melanoma diagnosed at stage III (n = 21) or IV (n = 19). The P-values were calculated using unpaired t test with Welch's correction for comparisons of normal skin versus melanoma stage III (P = 0.0421) and for normal skin versus melanoma stage IV (P = 0.0288).

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Fig. S4. Silencing of TRIB2 in melanoma cells activates FOXO dependent transcription. G-361FireFOX cells were transiently transfected with 2 different shRNAs against TRIB2 respectively or with shRNAs against PI3K or Akt, using Renilla luciferase as a transfection control. After 48 hours, firefly luciferase activity was measured and normalized against Renilla luciferase activity. The results refer to the average obtained in triplicate experiments and the error bars are representative of the standard deviation obtained in each triplicate. Statistical significance was determined by t-test (*, P> 0.05).