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1 Supplementary Materials for FOXP1 potentiates Wnt/β-catenin signaling in diffuse large B cell lymphoma Matthew P. Walker, Charles M. Stopford, Maria Cederlund, Fang Fang, Christopher Jahn, Alex D. Rabinowitz, Dennis Goldfarb, David M. Graham, Feng Yan, Allison M. Deal, Yuri Fedoriw, Kristy L. Richards, Ian J. Davis, Gilbert Weidinger, Blossom Damania, Michael B. Major* The PDF file includes: *Corresponding author. benmajor@med.unc.edu Published 3 February 2015, Sci. Signal. 8, ra12 (2015) DOI: /scisignal Fig. S1. FOXP1 promotes Wnt signaling in multiple cell types. Fig. S2. Isoform-specific effects of FOXP1 on β-catenin dependent transcription. Fig. S3. FOXP1 does not affect β-catenin dependent transcription through SOX17. Fig. S4. FOXP1 promotes the interaction between β-catenin and TCF7L2. Fig. S5. FOXP1 binds to the promoters of Wnt target genes. Fig. S6. FOXP1 alters β-catenin occupancy and histone acetylation. Fig. S7. TCF7L2 synergizes with FOXP1 to potentiate β-catenin dependent transcription. Fig. S8. FOXP1 abundance positively correlates with Wnt pathway activity in lymphoma cell lines. Fig. S9. Correlations between FOXP1 abundance and cellular responsiveness to Wnt antagonists and chemotherapeutics. Table S1. Top candidates from CDt/MS analysis. Table S2. sirnas used. Table S3. qpcr primers used. Table S4. ChIP primers used.

2 Supplementary Figure 1: FOXP1 promotes Wnt signaling in multiple cell types. (A- B) Quantification of BAR reporter expression in the indicated cell lines following FOXP1 overexpression. 24 hours post-transfection, cells were treated with control or Wnt3a conditioned media. (C) TOPFLASH reporter quantitation in KARPAS422 cells following control or FOXP1 overexpression. (D) Western blot of HT1080, HEK293T, HCT116, DB and KARPAS422 cell lines transfected with the indicated sirnas. (E,F) Quantification of β-catenin-dependent reporter expression in HT1080 cells and KARPAS422 cells after transfection with the indicated sirna in the presence and absence of Wnt3a conditioned media. (G) Quantification of AXIN2 and NKD1 mrna following FOXP1 overexpression in HT1080 cells. (H) Quantification of AXIN2 and NKD1 mrna following FOXP1 overexpression in RCK8 cells and Western blot showing FOXP1 expression. (I) Quantification of AXIN2 and NKD1 mrna following FOXP1 knockdown in KARPAS422 cells. (J) In shrna-control or shrna-foxp1 stable DB cells, FOXP1, AXIN2, and NKD1 gene expression was determined by qrt- PCR analysis. (K) Quantification of BAR activity in DB cells transfected with FOXP1 or control and treated with rhdkk1 (200 ng/ml) or C59 (200 nm). For (A-K), N=at least three independent biological experiments. For (A to C, E to K) significance was determined by Wilcoxon Signed Rank * < 0.05; **<0.005; ***< Error bars represent SEM.

3 Supplementary Figure 2: Isoform-specific effects of FOXP1 on β-catenin dependent transcription. (A) Several FOXP1 isoforms were cloned and tested for their ability to modulate Wnt signaling in 293T cells. Schematic of FOXP1 isoforms studied (15). (B) Quantification of BAR activity in HEK293T cells following transfection with FOXP1 isoforms. 24 hours post-transfection, cells were treated with control or Wnt3a conditioned media. Significance was determined by Wilcoxon Signed Rank across a minimum of three independent experiments. * < 0.05, **<0.005; error bars represent SEM. (C) Western blot analysis of HEK293T cells transfected with the indicated plasmid. (N=3 independent experiments) (D) Quantification of BAR activity in HEK293T cells transfected with murine Foxp1 or the murine ES-specific splice variant of Foxp1(Foxp1-ES). (N=3 independent experiments) (E) Western blot of analysis of indicated cell lines with FOXP1 or β-tubulin (TUBB) antibody. (N=3 independent experiments)

4 Supplementary Figure 3: FOXP1 does not affect β-catenin dependent transcription through SOX17. (A) SOX17 mrna expression was determined after transfection with control, SOX17-A, or SOX17-B sirna. (B) Quantification of BAR luciferase activity in HEK293T cells transfected with control or FOXP1 and control, SOX17-A, or SOX17-B sirna. (A-B) Graphs represent data obtained in biological triplicate.

5 Supplementary Figure 4: FOXP1 promotes the interaction between β-catenin and TCF7L2. HEK293T cells were transfected with pglue-β-catenin (70) and control or FOXP1 sirna. Streptavidin tagged β-catenin was affinity purified using streptavidin coupled sepharose beads. Co-complexed endogenous proteins were detected by Western blot. Right panel, biological triplicate experiments were quantified using LI-COR software. Significance was determined by Wilcoxon Signed Rank across biological triplicates. * < 0.05; Error bars represent SEM

6 Supplementary Figure 5: FOXP1 binds to the promoters of Wnt target genes. (A) ChIP analysis of stable knockdown DB cells using FOXP1 or β-catenin antibodies. Percent input was determined by qpcr. (B-C) ChIP analysis of AXIN2 or PDE4B in HCT116 cells treated with control or FOXP1 sirna. Control IgG, FOXP1, or β-catenin ChIP antibodies were used. (A-C) Error bars represent SEM for biological triplicates. Significance was determined by Wilcoxon Signed Rank.

7 Supplementary Figure 6: FOXP1 alters β-catenin occupancy and histone acetylation. (A-E) ChIP analysis of the indicated promoter in HCT116 cells following transfection with control or FOXP1 sirnas. The following ChIP antibodies were used: β-catenin (A,B), CBP (C,D), and histone H3-K9 acetylation (E). (A-E) Error bars represent SEM. Significance was determined by Wilcoxon Signed Rank across biological triplicates.

8 Supplementary Figure 7: TCF7L2 synergizes with FOXP1 to potentiate β-catenin dependent transcription. (A) Quantification of BAR activity in HEK293T cells transfected with increasing amounts of TCF7L2 in the presence or absence of FOXP1 overexpression. Below, Western blot analysis of TCF72, LEF1, FOXP1 (FLAG) and HC- RED (FLAG) as a control. Blots and graph are representative of data obtained in biological triplicate. (B) Quantification of BAR activity in HEK293T cells transfected with control or FOXP1, and sicnt, sitcf7l2-a, or sitcf7l2-b and treated as indicated. Graph is representative of data obtained in biological triplicate.

9 Supplementary Figure 8: FOXP1 abundance positively correlates with Wnt pathway activity in lymphoma cell lines. (A,B) C59-induced repression of AXIN2 (A) and NKD1 (B) gene expression was determined by qrt-pcr. Significance was determined by Wilcoxon Signed Rank across biological triplicates. NS not significant, * < 0.05 and **<0.005; error bars represent SEM.

10 Supplementary Figure 9: Correlations between FOXP1 abundance and cellular responsiveness to Wnt antagonists and chemotherapeutics. DB, KARPAS422, or RCK8 cell lines were treated with indicated chemotherapies and Wnt antagonists. Data was obtained in biological triplicate. Cell viability was determined by PrestoBlue quantitation.

11 Supplementary Table 1: Top candidates from CDt/MS analysis. Gene Gene ID COMPASS Fold Change Name Score FOXP EIF5A FOXP CTBP THBS FAM120A DSC1* KRT PFKFB IFI LGALS TXNRD SLC22A KRT TXNDC LGALS ANXA KRT MAGEA S100A9* Identified proteins were filtered for fold change relation to controls over 3 and ranked by the COMPASS WD score. *- indicates known Wnt pathway regulator. Combined results of 4 screens. Supplemental Table 2: sirnas used. Gene Name Sense Sequence CREBBP-A CAGACCCACCCAGGCCUCCUCAAUA CREBBP-B GGCGCAAGUCAUGAAUGGAUCUCUU CTNNB1 GGUGGUGGUUAAUAAGGCUTT FOXP1-A UGGCUAUGAUGACACCUCAAGUUAU FOXP1-B CAGCCCAAUGUAGAGUACAAAUGCA SOX17-A CCGACGCCAGCUCCGCGGUAUAUUA SOX17-B GGAGGUGGACCGCACGGAAUUUGAA

12 Supplemental Table 3: qpcr primers used. Gene FWD Primer Rev Primer Target AXIN2 GCGATCCTGTTAATCCTTATCAC AATTCCATCTACACTGCTGTC FOXP1 TCTTTGACGTGTACAGGATGCAC GCATACCAACACAACGAGTG NKD1 GGAGAGAGTGAGCGAACCCT CTTGCCGTTGTTGTCAAAGTC SOX17 TGGCTTTTCTTTATCTCCCAGCAG TTGCCCAGCATCTTGCTCAACTC GAPDH ATGGGGAAGGTGAAGGT AAGCTTCCCGTTCTCAG ACTB AGGCCAACCGCGAAGATGACC GAAGTCCAGGGCGACGTAGCAC Supplemental Table 4: ChIP primers used. Gene Target FWD Primer Rev Primer AXIN2 TGCTTGCCACTGTTTGAAGTCAGC GCCATGAACCCTTTTTGATCTTGC AXIN2 CTGGCTTTGGTGAACTGTTG AGTTGCTCACAGCCAAGACA ORF DNAJB1 TTTCTTAGTGGTGGGCTCTTCAGC TCAGGAGGCAGGAGGATGGTCT PDE4B TGGCTTTTCTTTATCTCCCAGCAG TGTCCCCTTTGAACTCTATCCAACA