Supplementary Information. Induction of p53-independent apoptosis by ectopic expression of HOXA5

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Supplementary Information Induction of p53-independent apoptosis by ectopic expression of in human liposarcomas Dhong Hyun Lee 1, *, Charles Forscher 1, Dolores Di Vizio 2, 3, and H. Phillip Koeffler 1, 4. 1 Division of Hematology and Oncology, 2 Division of Cancer Biology and Therapeutics, Departments of Surgery, Biomedical Sciences and Pathology and Laboratory Medicine, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, CA. 3 The Urological Diseases Research Center; Boston Children's Hospital, Boston, MA, Department of Surgery, Harvard Medical School, Boston, MA. 4 National Cancer Institute and Cancer Science Institute, National University of Singapore, Singapore. * Correspondence: Dhong Hyun Lee, Ph. D. Division of Hematology and Oncology, Cedars-Sinai Medical Center, UCLA School of Medicine, 8700 Beverly Blvd, D5022, Los Angeles, CA 90048, USA; Phone: +1-310-423-7736; Fax: +1-310-423-0225; E-mail: hiromasa@ucla.edu

0.0 0.2 0.4 0.6 0.8 1.0 Supplementary Figure S1 a p c D N A 3.1 -m ir -2 6 a -2 b a n ti-m ir -2 6 a -2 S W 8 7 2 S W 8 7 2 L P S 1 4 1 L P S 1 4 1 L P 6 L P 6 L P 1 L P 1 L P 2 L P 2 L P 3 L P 3 T 7 7 8 T 7 7 8 T 1 0 0 0 T 1 0 0 0 L IS A-2 L IS A-2 S A-4 S A-4 1 1 0 1 0 0 1 0 0 0 Log (R el E xp) R e l E x p Supplementary Figure S1. Validation of mir-26a-2 modulation in LPS cells. (a) For the overexpression study shown in Figure 1e, LPS cells were transfected with either mir-26a-2 expression vector or empty vector control (EV). After 48 h of transfection, cells were harvested and subjected to qrt-pcr. Data represent average relative mir-26a-2 expression ± standard deviation (SD, error bars). Dashed line indicates the expression level of mir-26a-2 in cells with EV. (b) For the inhibition study shown in Figure 1f, LPS cells were transfected with either anti-mir26a-2 or scrambled oligos (SCR). After 48 h, cells were harvested and subjected to qrt-pcr. Dashed line indicates the expression level of mir-26a-2 in cells with SCR.

Supplementary Figure S2 SW872 T778 LPS141 Flag- - + - + - + Flag CEBPA CEBPB PPARG GAPDH Supplementary Figure S2. Changes in the protein levels of key adipocyte differentiation proteins upon overexpression in LPS cells. LPS cells were transfected with either expression vector or empty vector control. Cells were harvested 24 h after transfection and subjected to Western blot analysis. Representative images are shown. GAPDH was used as a loading control.

PI EV Supplementary Figure S3 a T778 b 2 2 80 6 11 7 10 % Apoptotic Cells 60 40 20 NS NS NS 39 32 0 SW872 T778 LPS141 FITC-Annexin V DMSO Nec-1 EV+DMSO EV+Nec-1 +DMSO +Nec-1 Supplementary Figure S3. Effect of necrostatin-1 (Nec-1) on the -induced apoptosis in LPS cells. LPS cells were transfected with either expression vector or empty vector control (EV), and subsequently treated with 5 µm Nec-1 12h after transfection. Cells were further incubated for an additional 12h, and subjected to apoptosis assay. (a) Representative apoptosis assay results of T778 cells. Numbers indicate the percentage of early-apoptotic (bottom) and late-apoptotic (top) cells. (b) Summary of apoptosis assay results. Data represent % apoptotic cells ± standard deviation (SD, error bars). NS = not significant.

Supplementary Figure S4 T778 N C Flag- RELA HNRNPA1 TUBA4A - + + - + + CAPE - - + - - + 1.0 3.7 1.3 1.0 1.4 0.9 Supplementary Figure S4. Validation of caffeic acid phenethyl ether (CAPE) activity in LPS cells. T778 cells were transfected with either expression vector or empty vector control. Cells were treated with 25 µg/ml CAPE 9 h after transfection, incubated for an additional 15 h, and subjected to Western blotting. Representative images are shown. HNRNPA1 and TUBA4A (αtubulin) were used as loading control for nuclear (N) and cytoplasmic (C) fraction of cells, respectively. Numbers indicate relative band intensity of RELA normalized to the intensity of the genes in control (1.0) for each fraction.

R e l E x p R e l E x p R e l E x p Supplementary Figure S5 a F A S F A S L G b BAD a d 2.0 1.5 2.0 1.5 1.0 1.5 1.0 1.0 0.5 0.5 0.5 0.0 S W 8 7 2 T 7 7 8 L P S 1 4 1 0.0 N D N D N D S W 8 7 2 T 7 7 8 L P S 1 4 1 0.0 S W 8 7 2 T 7 7 8 L P S 1 4 1 Supplementary Figure S5. Effect of overexpression on the transcription of selected NFκB target genes in LPS cells. SW872, T778, LPS141 cells were transfected with either expression vector or empty vector control (EV). 24h after transfection, cells were harvested and subjected to qrt-pcr (same condition as shown in Figures 4f and 4g). Primer pairs are shown in Supplementary Table S2. Graphs show changes in the mrna expression level of selected proapoptotic (panel A) and anti-apoptotic (panel B) NFκB target genes upon overexpression in LPS cells. Dashed line indicates the expression level of each gene in cells with EV. Data represent relative mrna expression ± standard deviation (SD, error bars). ND = no detection.

PI EV % A p o p to tic C e lls Supplementary Figure S6 a T778 b 4 10 2 8 8 0 6 0 * * * 4 0 6 13 2 0 39 45 0 S W 8 7 2 T 7 7 8 L P S 1 4 1 SCR APC-Annexin V shrela-2 E V + S c r E V + s h R e la H O X A 5 + S c r H O X A 5 + s h R e la Supplementary Figure S6. Effect of RELA inhibition on the -induced apoptosis in T778 cells. T778 cells stably transfected with either shrna vector against RELA (shrela-2) or scrambled control (SCR), and were subsequently transfected with either expression vector () or empty vector control (EV). Cells were incubated 24 h and subjected to apoptosis assay analysis. The other shrela vector (shrela-1) was cytotoxic and was excluded from the study. (A) Representative apoptosis assay results. Numbers indicate the percentage of early-apoptotic (bottom) and late-apoptotic (top) cells. (B) Summary of apoptosis assay results. Data represent % apoptotic cells ± standard deviation (SD, error bars). Asterisk (*) indicates p-value less than 0.05 by t-test.

Supplementary Table S1. Human liposarcoma (LPS) cell lines used in the study Name LPS subtype Source TP53 status MDM2 status SW872 Undifferentiated ATCC c Mutant T356A (pi119n) WT LPS141 DDLPS a Dr. Fletcher d WT h Overexpressed LPS6 DDLPS Dr. Fletcher WT Overexpressed LPS1 DDLPS Dr. Wu e WT Overexpressed LPS2 DDLPS Dr. Wu WT Overexpressed LPS3 DDLPS Dr. Wu WT Overexpressed T778 WDLPS b Dr. Pedeutour f WT Overexpressed T1000 DDLPS Dr. Pedeutour WT Overexpressed LISA-2 Poorly differentiated Dr. Möller g Mutant T926C (pl309s) N/A SA-4 N/A N/A WT N/A Note: a DDLPS = Dedifferentiated LPS; b WDLPS = Well-differentiated LPS; c ATCC = American Type Culture Collection; d Dr. Christopher D. M. Fletcher at Brigham and Women s Hospital (Boston, MA) 1 ; e Dr. Hong Wu at University of California, Los Angeles (Los Angeles, CA) 2 ; f Dr. Florence Pedeutour at Nice University (Nice, France) 3 ; g Dr. Peter Möller at University of Ulm (Ulm, Germany) 4. h WT = Wild Type.

Supplementary Table S2. Primer sets used in this study Forward Primer Sequence Gene ID Reverse Primer Sequence For quantitative reverse-transcription real-time PCR (qrt-pcr) GAPDH 5 -CAGCAAGAGCACAAGAGGAA-3 5 -TCTACATGGCAACTGTGAGGAG-3 5 -CCAGATCTACCCCTGGATG-3 5 -ACTTCATTCTCCGGTTTTGG-3 TP53 5 -CCCAAGCAATGGATGATTTGA-3 5 -GGCATTCTGGGAGCTTCATCT-3 FASLG 5 -TCTACCAGCCAGATGCACAC-3 5 -TCACTCCAGAAAGCAGGACA-3 FAS 5 -TCAGTACGGAGTTGGGGAAG-3 5 -CCAATCCCTTGGAGTTGATG-3 BAD 5 -GCTGACCCAGATTCCCTTC-3 5 -TAAACCTGGCTCGCGACTT-3 For cloning 3 UTR-WT a 3 -UTR-Mut b CDS c 5 -GCCGTGTAATTCTAGAGTTCTCGTTGCCCTAA TTCATC-3 5 -CCGCCCCGACTCTAGAAGGAACACTTCCACGC ACA-3 5'-AATAGATGTTTTAACTTATTTATATGAAGCAA GCTGTGTTCATTTAGGTAACTATAACAAAAAAAGAAA AGAGAAAAAAAAACACAC-3' 5'-GTGTGTTTTTTTTTCTCTTTTCTTTTTTTGTT ATAGTTACCTAAATGAACACAGCTTGCTTCATATAAA TAAGTTAAAACATCTATT-3' 5 -CGGAATTCATGAGCTCTTATTTTGTAAACTC-3 5 -CGGAATTCTCAGGGACGGAAGGCCCCTCCT-3 Note Loading control pgl3 pgl3 pcdna3.1-flag Note: a WT = wild type; b Seed sequence of mir-26a-2 binding site (UACUUGAA) was mutated to UCAUUUAG by site-directed mutagenesis; c CDS = coding sequence

Supplementary Table S3. Antibodies used in this study Antibody Phosphorylation Site Company Catalog No For Western blotting GAPDH Santa Cruz sc-47724 Flag Sigma F3165 phospho-p53 Ser15 Cell Signaling 9284 p53 Cell Signaling 2524 PARP Cell Signaling 9532 CASP3 Cell Signaling 9668 CASP8 Santa Cruz sc-7890 CASP9 Santa Cruz sc-7885 phospho-rela Ser536 Cell Signaling 3033 RELA Santa Cruz sc-372 NFKBIA Santa Cruz sc-847 (IκBα) HNRNPA1 Santa Cruz sc-10030 TUBA4A Santa Cruz sc-5286 (α-tubulin) FOS Santa Cruz sc-52 TNFR1 R & D Systems MAB225 TNFR2 Santa Cruz sc-8041 BAX Santa Cruz sc-493 BCL2 Santa Cruz 7382 Bcl-xL Cell Signaling 2762 CEBPA Santa Cruz sc-9315 CEBPB Santa Cruz sc-150 PPARG Cell Signaling 2243 For Immunocytochemistry RELA Cell Signaling 6956 FITC-IgG Abnova PAB4971

REFERENCES TO SUPPLEMENTARY INFORMATION 1 Snyder, E. L. et al. c-jun amplification and overexpression are oncogenic in liposarcoma but not always sufficient to inhibit the adipocytic differentiation programme. J Pathol 218, 292-300, doi:10.1002/path.2564 (2009). 2 Smith, K. B. et al. Novel dedifferentiated liposarcoma xenograft models reveal PTEN down-regulation as a malignant signature and response to PI3K pathway inhibition. Am J Pathol 182, 1400-1411, doi:10.1016/j.ajpath.2013.01.002 (2013). 3 Pedeutour, F. et al. Structure of the supernumerary ring and giant rod chromosomes in adipose tissue tumors. Genes Chromosomes Cancer 24, 30-41 (1999). 4 Wabitsch, M. et al. LiSa-2, a novel human liposarcoma cell line with a high capacity for terminal adipose differentiation. Int J Cancer 88, 889-894 (2000).

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