Legends to Supplementary Material

Legends to Supplementary Material

Supplementary Figure 1. Comparison of FoxO target gene expression levels in lymphoma loss-of- and fibroblast gain-of-function model systems. (A) Validation of selected genes (drebrin 1 [Dbn1], reticulon 3 [Rtn3], YY1 transcription factor [YY1], and tissue inhibitor of metalloproteinase [Timp3]) by RQ-PCR whose expression levels were found to be substantially altered by microarray analysis in dnFoxO-lymphomas as compared to those without the dnFoxO moiety (n = 6 each). Values are plotted as the relative fold change (– dnFoxO taken as reference). (B) RQ-PCR-based time course analysis of the same panel of transcripts as in A in 3T3-Foxo3aA3-ER cells after addition of 4-OHT. Note that all gene products with lower expression levels in dnFoxO-driven lymphomas display strong inducibility in response to FoxO3A activation (Dbn1, Rtn3, YY1), and, likewise, appear to be repressed following FoxO3A when induced in the presence of dnFoxO (Timp3).

Supplementary Figure 2. Expression of growth regulators in individual Myc-driven lymphomas with and without dnFoxO. (A) Expression analysis of p53 and dnFoxO transcripts by RT-PCR in another 16 (i.e. samples 13-28; cf. Fig.1D) individual lymphomas of the indicated genotypes with S16 as control. (B) RQ-PCR documenting expression of Bim mRNA in the lymphoma samples shown in Fig. 2A. RNA was extracted from the indicated lymphoma samples and amplified with a primer set specific for Bim; the primers amplify mRNA encoding all splice variants. Data are plotted as ∆Ct values relative to a control mRNA (encoding ribosomal protein s16). (C) RQ-PCR analyses of eIF4E and 4EBP1 mRNAs; samples as in B. (D) Immunoblot analyses documenting expression levels and phosphorylation status of the indicated proteins in individual lymphomas; samples as in B. Please note that the anti-phospho-mTOR antibodies were able to detect a positive control (data not shown). (E) Genomic multiplex PCR analysis of the indicated exons of the Ink4a/Arf locus; samples are as in B (Arf-/- and Ink4a/Arf+/- MEFs as controls).

Supplementary Figure 3. The dnFoxO moiety reduces Arf expression and protects from genomic Arf deletions during Eµ-myc;Arf+/- lymphomagenesis. (A) Multiplex PCR analysis of the indicated exons of the Ink4a/Arf locus based on genomic DNA isolates from lymphomas that formed in lethally irradiated recipients upon receiving Eµ-myc;Arf+/- fetal liver cells stably transduced with a retrovirus encoding dnFoxO-IRES-GFP (lymphomas were considered “dnFoxO +” if GFP-positive [n = 4; three cases shown] and “dnFoxO –“ if GFP-negative [n = 3; one case shown] as assessed by flow cytometry [data not shown]; Ink4a/Arf-null and p53-null lymphomas as controls). (B) Representative fluorescence microscopy photomicrographs from lymphoma cells as in A as well as control Eµ-myc lymphoma cells showing p19Arf immunofluorescence (left panel), GFP fluorescence (center panel), and DAPI as nuclear counterstain (right panel). Note that the Arf+/-;-dnFoxO lymphoma has deleted the remaining Arf allele and is therefore Arf-null. (C) Expression analysis of Arf and TATA-box binding protein (TBP; as an internal control) transcripts by RT-PCR in individual lymphomas as in A. Note the much lower (compared to control and p53-null lymphomas) and very low signals in all Arf+/-;+dnFoxO cases [n = 4; three cases shown], but the virtually undetectable signal in the Arf+/-;-dnFoxO case [n = 3; one case shown] due to the bi-allelic deletion of exon 1b, as shown in B.

Supplementary Figure 4. FoxO action in fibroblast model systems. (A) Detection of Myc and dnFoxO transcripts (with s16 as an internal control) by RT-PCR; MEFs were infected with retroviruses expressing the indicated proteins, and RNA was isolated from pools of infected cells immediately after selection with puromycin. (B) The MEF population generated on a 3T3 protocol to be engineered as 3T3-Foxo3aA3-ER cells expresses p19Arf and displays an induction of p53 protein expression 6 hours after addition of 1 µg/ml of ADR, as demonstrated by immunoblot analysis with a-tubulin as a loading control. (C) 3T3 MEFs express detectable amounts of FoxO3aA3-ER protein after stable transduction as evidenced by immunoblot analysis against the ER component with Cdk2 as a loading control. (D) Arf mRNA expression (relative to S16 mRNA) by RQ-PCR in 3T3-FoxO3aA3-ER cells at the indicated time points after activation of FoxO3aA3-ER by addition of 4-OHT with and without pre-treatment with cycloheximide 10 minutes before.

Supplementary Table 1. Summary of microarray experiments. All 12 lymphomas shown in Fig. 1D and Fig. 2 were subjected to a microarray analysis. The table summarizes the expression of the six genes that discriminate most clearly the six tumors expressing dnFoxO from those that do not express dnFoxO and the average expression difference between the two groups. The table also shows the fold regulation of the same genes in the FoxO3aA3-ER cell line described in the text. Supplementary Fig.1 shows RQ-PCR data from the lymphomas and a detailed time course to validate these results for four of the genes.