Supplementary Information. Induction of p53-independent apoptosis by ectopic expression of HOXA5
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1 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: ; Fax: ; hiromasa@ucla.edu
2 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 S W L P S L P S 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 T T T L IS A-2 L IS A-2 S A-4 S A 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.
3 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.
4 PI EV Supplementary Figure S3 a T778 b % Apoptotic Cells NS NS NS 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.
5 Supplementary Figure S4 T778 N C Flag- RELA HNRNPA1 TUBA4A CAPE 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.
6 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 S W T L P S N D N D N D S W T L P S S W T L P S 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.
7 PI EV % A p o p to tic C e lls Supplementary Figure S6 a T778 b * * * S W T L P S 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.
8 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.
9 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
10 Supplementary Table S3. Antibodies used in this study Antibody Phosphorylation Site Company Catalog No For Western blotting GAPDH Santa Cruz sc 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 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
11 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, , doi: /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, , doi: /j.ajpath (2013). 3 Pedeutour, F. et al. Structure of the supernumerary ring and giant rod chromosomes in adipose tissue tumors. Genes Chromosomes Cancer 24, (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, (2000).
Induction of p53-independent apoptosis by ectopic expression of HOXA5 in human liposarcomas
Induction of p53independent apoptosis by ectopic expression of HOXA5 in human liposarcomas The Harvard community has made this article openly available. Please share how this access benefits you. Your
Supplementary Material
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Supplemental Table S1
Supplemental Table S. Tumorigenicity and metastatic potential of 44SQ cell subpopulations a Tumorigenicity b Average tumor volume (mm ) c Lung metastasis d CD high /4 8. 8/ CD low /4 6./ a Mice were injected
A Normal Exencephaly Craniora- Spina bifida Microcephaly chischisis. Midbrain Forebrain/ Forebrain/ Hindbrain Spinal cord Hindbrain Hindbrain
A Normal Exencephaly Craniora- Spina bifida Microcephaly chischisis NTD Number of embryos % among NTD Embryos Exencephaly 52 74.3% Craniorachischisis 6 8.6% Spina bifida 5 7.1% Microcephaly 7 1% B Normal
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Supplementary Figure 1
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Relative expression ERE-luciferase activity activity (pmole/min) activity (pmole/min) activity (pmole/min) activity (pmole/min) MCF-7 KPL-3C ZR--1 BT-474 T47D HCC15 KPL-1 HBC4 activity (pmole/min) a d
SUPPLEMENTAL FIGURE LEGENDS
SUPPLEMENTAL FIGURE LEGENDS Supplemental Figure S1: Endogenous interaction between RNF2 and H2AX: Whole cell extracts from 293T were subjected to immunoprecipitation with anti-rnf2 or anti-γ-h2ax antibodies
Tbk1-TKO! DN cells (%)! 15! 10!
a! T Cells! TKO! B Cells! TKO! b! CD4! 8.9 85.2 3.4 2.88 CD8! Tbk1-TKO! 1.1 84.8 2.51 2.54 c! DN cells (%)! 4 3 2 1 DP cells (%)! 9 8 7 6 CD4 + SP cells (%)! 5 4 3 2 1 5 TKO! TKO! TKO! TKO! 15 1 5 CD8
Supplementary Figure 1. HOPX is hypermethylated in NPC. (a) Methylation levels of HOPX in Normal (n = 24) and NPC (n = 24) tissues from the
Supplementary Figure 1. HOPX is hypermethylated in NPC. (a) Methylation levels of HOPX in Normal (n = 24) and NPC (n = 24) tissues from the genome-wide methylation microarray data. Mean ± s.d.; Student
Supplementary Figure S I: Effects of D4F on body weight and serum lipids in apoe -/- mice.
Supplementary Figures: Supplementary Figure S I: Effects of D4F on body weight and serum lipids in apoe -/- mice. Male apoe -/- mice were fed a high-fat diet for 8 weeks, and given PBS (model group) or
Supplementary Figure 1: Expression of NFAT proteins in Nfat2-deleted B cells (a+b) Protein expression of NFAT2 (a) and NFAT1 (b) in isolated splenic
Supplementary Figure 1: Expression of NFAT proteins in Nfat2-deleted B cells (a+b) Protein expression of NFAT2 (a) and NFAT1 (b) in isolated splenic B cells from WT Nfat2 +/+, TCL1 Nfat2 +/+ and TCL1 Nfat2
Supplementary Materials for
www.sciencesignaling.org/cgi/content/full/9/439/ra78/dc1 Supplementary Materials for Small heterodimer partner mediates liver X receptor (LXR) dependent suppression of inflammatory signaling by promoting
Supplementary Figure 1. A. Bar graph representing the expression levels of the 19 indicated genes in the microarrays analyses comparing human lung
Supplementary Figure 1. A. Bar graph representing the expression levels of the 19 indicated genes in the microarrays analyses comparing human lung immortalized broncho-epithelial cells (AALE cells) expressing
Supplementary Material
Supplementary Material accompanying the manuscript Interleukin 37 is a fundamental inhibitor of innate immunity Marcel F Nold, Claudia A Nold-Petry, Jarod A Zepp, Brent E Palmer, Philip Bufler & Charles
Supplementary Materials for
www.sciencesignaling.org/cgi/content/full/2/1/ra81/dc1 Supplementary Materials for Delivery of MicroRNA-126 by Apoptotic Bodies Induces CXCL12- Dependent Vascular Protection Alma Zernecke,* Kiril Bidzhekov,
Tumor suppressor Spred2 interaction with LC3 promotes autophagosome maturation and induces autophagy-dependent cell death
www.impactjournals.com/oncotarget/ Oncotarget, Supplementary Materials 2016 Tumor suppressor Spred2 interaction with LC3 promotes autophagosome maturation and induces autophagy-dependent cell death Supplementary
% of Cells A B C. Proliferation Index. T cell count (10 6 ) Division Index. % of Max CFSE. %Ki67+ cells. Supplementary Figure 1.
A B C T cell count (1 6 ) 3. 2. 1.. * % of Max CFSE ormoxia ypoxia o Stim. Proliferation Index 2.5 2. 1.5 1. * Division Index 2. 1.5 1..5. D E %Ki67+ cells 1 8 6 4 2 % of Cells 8 ormoxia 6 ypoxia 4 2 *
TRAF6 ubiquitinates TGFβ type I receptor to promote its cleavage and nuclear translocation in cancer
Supplementary Information TRAF6 ubiquitinates TGFβ type I receptor to promote its cleavage and nuclear translocation in cancer Yabing Mu, Reshma Sundar, Noopur Thakur, Maria Ekman, Shyam Kumar Gudey, Mariya
Supplementary Figure 1
Combination index (CI) Supplementary Figure 1 2. 1.5 1. Ishikawa AN3CA Nou-1 Hec-18.5...2.4.6.8 1. Fraction affected (Fa) Supplementary Figure 1. The synergistic effect of PARP inhibitor and PI3K inhibitor
Supplementary Figure 1. The CagA-dependent wound healing or transwell migration of gastric cancer cell. AGS cells transfected with vector control or
Supplementary Figure 1. The CagA-dependent wound healing or transwell migration of gastric cancer cell. AGS cells transfected with vector control or 3xflag-CagA expression vector were wounded using a pipette
Supplementary Figure 1: si-craf but not si-braf sensitizes tumor cells to radiation.
Supplementary Figure 1: si-craf but not si-braf sensitizes tumor cells to radiation. (a) Embryonic fibroblasts isolated from wildtype (WT), BRAF -/-, or CRAF -/- mice were irradiated (6 Gy) and DNA damage
Supplementary Fig. 1 eif6 +/- mice show a reduction in white adipose tissue, blood lipids and normal glycogen synthesis. The cohort of the original
Supplementary Fig. 1 eif6 +/- mice show a reduction in white adipose tissue, blood lipids and normal glycogen synthesis. The cohort of the original phenotypic screening was n=40. For specific tests, the
Supplementary Figure 1 Cell line TRIB2 status. Supplementary Figure 2 TRIB2 status has no impact on the cell cycle after PI3K inhibition. a. b.
Supplementary Figure 1 Cell line TRIB2 status. TRIB2 protein expression to determine endogenous expression and to determine the effectiveness of each of our TRIB2 knockdown constructs. Supplementary Figure
SUPPLEMENTARY FIGURE LEGENDS
SUPPLEMENTARY FIGURE LEGENDS Supplementary Figure 1. Hippocampal sections from new-born Pten+/+ and PtenFV/FV pups were stained with haematoxylin and eosin (H&E) and were imaged at (a) low and (b) high
SUPPLEMENTARY INFORMATION. Supp. Fig. 1. Autoimmunity. Tolerance APC APC. T cell. T cell. doi: /nature06253 ICOS ICOS TCR CD28 TCR CD28
Supp. Fig. 1 a APC b APC ICOS ICOS TCR CD28 mir P TCR CD28 P T cell Tolerance Roquin WT SG Icos mrna T cell Autoimmunity Roquin M199R SG Icos mrna www.nature.com/nature 1 Supp. Fig. 2 CD4 + CD44 low CD4
Supplementary Figure S1 Expression of mir-181b in EOC (A) Kaplan-Meier
Supplementary Figure S1 Expression of mir-181b in EOC (A) Kaplan-Meier curves for progression-free survival (PFS) and overall survival (OS) in a cohort of patients (N=52) with stage III primary ovarian
Figure S1. Analysis of genomic and cdna sequences of the targeted regions in WT-KI and
Figure S1. Analysis of genomic and sequences of the targeted regions in and indicated mutant KI cells, with WT and corresponding mutant sequences underlined. (A) cells; (B) K21E-KI cells; (C) D33A-KI cells;
Supplementary Figures
Supplementary Figures Supplementary Figure 1 Increased ABHD5 expression in human colon cancer associated macrophages. (a) Murine peritoneal macrophages were treated with regular culture medium (Ctrl) or
SUPPLEMENTARY INFORMATION
DOI:.38/ncb3399 a b c d FSP DAPI 5mm mm 5mm 5mm e Correspond to melanoma in-situ Figure a DCT FSP- f MITF mm mm MlanaA melanoma in-situ DCT 5mm FSP- mm mm mm mm mm g melanoma in-situ MITF MlanaA mm mm
TITLE: MiR-146-SIAH2-AR Signaling in Castration-Resistant Prostate Cancer
AWARD NUMBER: W81XWH-14-1-0387 TITLE: MiR-146-SIAH2-AR Signaling in Castration-Resistant Prostate Cancer PRINCIPAL INVESTIGATOR: Dr. Goberdhan Dimri, PhD CONTRACTING ORGANIZATION: George Washington University,
Tel: ; Fax: ;
Tel.: +98 216 696 9291; Fax: +98 216 696 9291; E-mail: mrasadeghi@pasteur.ac.ir Tel: +98 916 113 7679; Fax: +98 613 333 6380; E-mail: abakhshi_e@ajums.ac.ir A Soluble Chromatin-bound MOI 0 1 5 0 1 5 HDAC2
Supplementary figure 2. VCAM1 protein expression in static HCAECs exposed to CSE over 72 hours, with TNFα or TNFα + CSE positive controls.
Supplementary data Cigarette smoke extract profoundly suppresses TNFα-mediated proinflammatory gene expression through upregulation of ATF3 in human coronary artery endothelial cells Jack E. Teasdale 1,
Supplementary Figure 1. MAT IIα is Acetylated at Lysine 81.
IP: Flag a Mascot PTM Modified Mass Error Position Gene Names Score Score Sequence m/z [ppm] 81 MAT2A;AMS2;MATA2 35.6 137.28 _AAVDYQK(ac)VVR_ 595.83-2.28 b Pre-immu After-immu Flag- WT K81R WT K81R / Flag
EPIGENETIC RE-EXPRESSION OF HIF-2α SUPPRESSES SOFT TISSUE SARCOMA GROWTH
EPIGENETIC RE-EXPRESSION OF HIF-2α SUPPRESSES SOFT TISSUE SARCOMA GROWTH Supplementary Figure 1. Supplementary Figure 1. Characterization of KP and KPH2 autochthonous UPS tumors. a) Genotyping of KPH2
Supplemental Information. Menin Deficiency Leads to Depressive-like. Behaviors in Mice by Modulating. Astrocyte-Mediated Neuroinflammation
Neuron, Volume 100 Supplemental Information Menin Deficiency Leads to Depressive-like Behaviors in Mice by Modulating Astrocyte-Mediated Neuroinflammation Lige Leng, Kai Zhuang, Zeyue Liu, Changquan Huang,
Liposarcoma*Genome*Project*
LiposarcomaGenomeProject July2015! Submittedby: JohnMullen,MD EdwinChoy,MD,PhD GregoryCote,MD,PhD G.PeturNielsen,MD BradBernstein,MD,PhD Liposarcoma Background Liposarcoma is the most common soft tissue
The p53 protein induces apoptosis, cell cycle arrest, senescence,
SCO2 Induces p53-mediated Apoptosis by Thr 845 Phosphorylation of ASK-1 and Dissociation of the ASK-1 Trx Complex Esha Madan, a,b Rajan Gogna, b Periannan Kuppusamy, c Madan Bhatt, d Abbas Ali Mahdi, a