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DD2 suppresses tumorigenicity of ovarian cancer cells by limiting cancer stem cell population Chunhua Han et al. Supplementary Materials and Methods Analysis of publicly available datasets: To analyze DD2 mrna expression in ovarian serous adenocarcinoma, we obtained the data from TCGA, Nature 211 by using www.cbioportal.org. Specifically, on the home page of the website, select download data, then, select Ovarian Serous Cystadenocarcinoma (TCGA, Nature 211), click mrna expression Z-score (all genes) from Select Genomic Profiles, and enter gene set DD2, select Transpose data matrix. Click Submit, the DD2 mrna Z- scores of 489 cases will appear. These Z-scores were then plotted and analyzed by using Minitab. To analyze the effect of DD2 expression on prognostic of ovarian cancer patients, we generated Kaplan-Meier survival curve of ovarian cancer patients with low or high expression of DD2 by using Kaplan-Meier Plotter (www.kmplot.com/analysis) (Fig. 6A,) and cioportal for Cancer Genomics (www.cbioportal.org) (Fig. C,D). Specifically, to generate Fig. 6A,, select Ovarian on the upper right corner of the home page of www.kmplot.com/analysis, input DD2 into Affy id/gene symbol, click Auto select best cutoff, then click Draw Kaplan- Meier plot. To generate Fig. 6C,D, select Query on the home page of the website www.cbioportal.org, select Ovarian Serous Cystadenocarcinoma (TCGA, Nature 211) from Select Cancer Study. In the Select Genomic Profiles, only select mrna expression Z-scores (all genes). In Enter Gene set, input DD2: Exp < -2, or DD2: Exp >.5, then click Submit. Click Survival tab, overall survival Kaplan-Meier Estimate will appear. To analyze the relationship between DD2 and Iκα mrna in ovarian cancers, we obtained the data from TCGA, Nature 211 by using www.cbioportal.org. Specifically, on the home page of the website, select download data, then, select Ovarian Serous Cystadenocarcinoma (TCGA, Nature 211), click mrna expression Z-score (all genes) from Select Genomic Profiles, and enter gene set DD2, NFK1A, select Transpose data matrix. Click Submit, the DD2 and NFK1A (encoding Iκα) mrna Z-scores of 489 cases will appear. The correlation between these Z-scores of two genes was then analyzed by Pearson correlation and plotted using Minitab. The DD2 mrna expression data, DD2 promoter methylation data, and DD2 copy number alteration (CNA) data in ovarian cancer patients were retrieved from TCGA dataset (Nature 211) by using a tool in www.cbioportal.org. Specifically, select Query on the home page of the website www.cbioportal.org, select Ovarian Serous Cystadenocarcinoma (TCGA, Nature 211) from Select Cancer Study. In the Select Genomic Profiles, select Putative copy-number alterations (GISTIC) and mrna expression Z-scores (all genes). In Enter Gene set, input DD2, then click Submit. On the next page, click Plots tab. From Plot Type, select mrna vs. Copy Number, the corresponding figure will show on the right. Select mrna vs. DNA Methylation, the corresponding figure will appear on the right.

Detection of SP cells using Flow cytometry. Ovarian cancer cell suspensions were stained with 5 µg/ml of Hoechst 33342 dye for 9 min at 37 C, washed, and resuspended in PS. efore cell sorting, 2 µg/ml propidium iodide was added for dead cell discrimination. Each cell lines included a tube with Verapamil (2µM) to serve as a control. SP cells were identified and electronically gated on Aria III Flow cytometer after excitation of the Hoechst dye with 15 mw of 35 nm UV light. SP fluorescence emissions were directed toward a 61-nm dichroic filter and captured simultaneously through both a blue (45-nm) band-pass and a red (675-nm) long-pass filter on a linearly amplified fluorescence scale. Primers for real-time RT-PCR analysis. Nanog- forward, 5 - GTC CCA AAG GCA AAC AAC CC -3, reverse, 5 - TTG ACC GGG ACC TTG TCT TC -3 ; Oct4A- forward, 5 -TCG CAA GCC CTC ATT TCA CC -3, reverse, 5 - CGA GAA GGC GAA ATC CGA AG -3 ; Sox2- forward, 5 - TCA GGA GTT GTC AAG GCA GAG -3, reverse, 5 - GGC AGC AAA CTA CTT TCC CC -3 ; E-Cadherin- forward, 5 - TGC CCA GAA AAT GAA AAA GG -3, reverse, 5 - GTG TAT GTG GCA ATG CGT TC -3 ; EpCAM- forward, 5 - CAG TTG GTG CAC AAA ATA CTG TC -3, reverse, 5 - CTC TCA TCG CAG TCA GGA TC -3 ; GAPDH- forward, 5 - GAA GGT GAA GGT CGG AGT -3, reverse, 5 - GAA GAT GGT GAT GGG ATT TC -3.

Supplementary Figures A A278 CP7 CP7-DD2-1 CP7-DD2-3 DD2 E-Cadherin Vimentin Tubulin CP7 CP7-DD2-1 CP7-DD2-3 C 28-shDD2-clone DD2 1 2 3 4 5 6 7 8 E-Cadherin Vimentin Actin Supplementary Figure S1: Manipulation of DD2 expression does not change EMT of ovarian cancer cells. A, protein extracts from ovarian cancer cell line A278, its derivative cisplatinresistant cell line CP7, and CP7 cells stably transfected with DD2-expressing vectors were immunoblotted for the expression of epithelial marker E-Cadherin and mesenchymal marker Vimentin., the cellular morphology of CP7 cells with or without DD2 overexpression. C, protein extracts from various clones selected from 28 cells stably transfected with shdd2 vectors were immunoblotted for the expression of E-Cadherin and Vimentin, as well as DD2.

DEA ALDEFLUOR 19.3% CP7-Vector 25 4.6% CP7-DD2-1 ALDH + cells (%) 2 15 1 5 * ** 3.8% CP7-DD2-3 CP7-Vector CP7-DD2-1 CP7-DD2-3 ALDEFLUOR Supplementary Figure S2: DD2 reduces the abundance of ALDH + cells in ovarian cancer cell lines. A, ALDEFLUOR staining of CP7-Vector and two DD2-overexpressing CP7 cell lines were repeated and analyzed by FACS with optimized channel settings on a LSRII Flow Cytometer. Number: percentage of ALDH + cells., average percentage of ALDH + cells in indicated cells.

278 CP7 CP7-DD2-1 CP7-DD2-3 DD2 ALDH1A1 Tubulin Supplementary Figure S3: Protein extracts from ovarian cancer cell line A278, its derivative cisplatin-resistant cell line CP7, and CP7 cells stably transfected with DD2-expressing vectors were immunoblotted for the expression of DD2 and ALDH1A1.

25 2 15 1 5 DEA 25 2 15 1 5 ALDEFLUOR.13% 28-shCtrl 25 2 15 1 5 25 2 15 1 5 2.29% 28-shDD2-3 SSC-A X 1 25 2 15 1 5 25 2 15 1 5 1.49% 28-shDD2-6 ALDEFLUOR 3.5 ** ALDH + cells (%) 3. 2.5 2. 1.5 1..5 **. 28-shCtrl 28-shDD2-3 28-shDD2-6 Supplementary Figure S4: A, ALDH activity was analyzed in 28 cells stably transfected with either control or DD2 shrna plasmids by FACS. Number: percentage of ALDH + cells., average percentage of ALDH + cells in indicated cells. ar: SD, n=3, **: P<.1

CD117 CD44 + CD117 + cells (%) 1 1 1 2 1 3 1 4 1 5.25.2.15.1.5. CD44.8% 1 1 1 2 1 3 1 4 1 5 1 1 1 2 1 3 1 4 1 5 CP7-Vector.21%.8% * CP7-DD2-1 CP7-Vector CP7-DD2-1 CP7-DD2-3 * CP7-DD2-3 C D CD44 + CD117 + cells (%) CD117 2. 1.5 1..5..82% 1 1 1 2 1 3 1 4 1 5 1.5% 1 1 1 2 1 3 1 4 1 5 1.12% 1 1 1 2 1 3 1 4 1 5 CD44 28-shCtrl * 28-shDD2-3 28-shDD2-6 28-shCtrl 28-shDD2-3 28-shDD2-6 Supplementary Figure S5: Overexpression of DD2 reduces, while knockdown of DD2 increases the percentage of CD44 + CD117 + cells in ovarian cancer cell lines. CD44 + CD117 + cells were analyzed in CP7-Vector and two DD2-overexpressing CP7 cell lines (A, ), as well as 28 cells stably transfected with either control or DD2 shrna plasmids (C, D) by FACS. ar: SD, n=3, *: P<.5

+ Verapamil.%.7% CP7-Vector.8.6.%.6% CP7-DD2-1 SP (%).4.2 *.%.2% CP7-DD2-3. CP7-Vector CP7-DD2-1 CP7-DD2-3 Supplementary Figure S6: Analysis of side population (SP) cells in CP7 and DD2 overexpressed CP7 cells. A, CP7 and DD2-overexpressed CP7 cells were labeled with Hoechst 33342 dye and analyzed by flow cytometry before and after treatment with Verapamil., average percentage of SP cells in indicated cell lines. ar: SD, n=3, *: P<.5

DEA ALDEFLUOR 25 25 SSC-A X 1 2 15 1 5 2 15 1 5 ALDEFLUOR Sphere formation rate (%) 1 ** 8 6 4 2 CP7-ALDH- CP7-ALDH+ C Cell type Cell dose Tumor formation CP7-ALDH + 1 4/4 1, 4/4 1, 4/4 CP7-ALDH - 1 /4 1, /4 1, 2/4 Supplementary Figure S7: Isolation and characterization of CP7-ALDH + cells. A, CP7 cells were stained with ALDEFLUOR and sorted with FACS. CP7-ALDH + cells were cultured in Ultra-Low attachment plates in serum-free DMEM/F12 medium supplemented with serum replacement, EGF and bfgf., Sphere formation of CP7-ALDH - and CP7-ALDH + cells was assessed as described in Materials and Methods. ar: SD, n=3, *: P<.5. C, Various amount of cells were mixed with Matrigel (1:1) and injected into nude mice subcutaneously. The formation of tumor was observed up to 3 months.

D SKOV3-sphere Relative Transcript Level 3 25 2 15 1 Cell type Cell dose Tumor formation 5 SKOV3-Adherent SKOV3-Spheroid ** * * Nanog Oct4 Sox2 C Sphere formation rate (%) 1 ** 8 6 4 2 SKOV3-Spheroid SKOV3-Adherent SKOV3- spheroid 1 3/4 1, 4/4 1, 4/4 SKOV3- adherent 1 /4 1, /4 1, /4 Supplementary Figure S8: Isolation and characterization of SKOV3-CSCs. A, SKOV3- spheroid was selected by culturing cells in Ultra-Low attachment 96-well plate in serum-free DMEM/F12 medium supplemented with serum replacement, EGF and bfgf., RNA was extracted from SKOV3-Adherent and SKOV3-Spheroid cultured cells, and various stem cell markers were determined using real-time RT-PCR. ar: SD, n=3, *: P<.5. C, Sphere formation of SKOV3-Adherent and SKOV3-Spheroid cultured cells was assessed as described in Materials and Methods. ar: SD, n=3, **: P<.1. D, Various amount of cells were mixed with Matrigel (1:1) and injected into nude mice subcutaneously. The formation of tumor was observed up to 3 months.

E Relative Transcript Level (%) A CD117 1 8 6 4 2 CD44 28 1.% 1 1 1 2 1 3 1 4 1 5 ** 28 28-CD44 + CD117 + ** ** ** Nanog Sox2 Oct4 E-Cadherin EpCAM C D CD117 F Relative Transcript Level (%) 12 1 8 6 4 2 28C13 1.5% 1 1 1 2 1 3 1 4 1 5 CD44 ** 28C13 Nanog Sox2 Oct4 E-Cadherin EpCAM Sphere formation rate (%) 1 28C13-CD44 + CD117 + * ** ** 8 6 4 2 G ** 28-Unsorted 28-CD44+CD117+ Cell types Sphere formation rate (%) 1 C13-Unsorted C13-CD44+CD117+ Cell dose Tumor formation 28-CD44 + CD117 + 1 4/4 1, 4/4 1, 4/4 Unsorted 28 1 /4 1, /4 1, /4 28C13- CD44 + CD117 + 1 3/4 1, 4/4 1, 4/4 Unsorted 28C13 1 /4 1, /4 1, /4 8 6 4 2 ** Supplementary Figure S9: Isolation and characterization of 28-CD44 + CD117 + and 28C13- CD44 + CD117 + cells. A,, 28 and 28C13 cells were stained with anti-cd44-fitc and anti- CD117-PE, and sorted with FACS. 28-CD44 + CD117 + and 28C13-CD44 + CD117 + cells were cultured in Ultra-Low attachment plates in serum-free DMEM/F12 medium supplemented with serum replacement, EGF and bfgf. C,D, Sphere formation of 28-CD44 + CD117 + and 28C13-CD44 + CD117 + cells was assessed as described in Materials and Methods. ar: SD, n=3, **: P<.1. E,F, RNA was isolated from unsorted 28, 28-CD44 + CD117 +, as well as unsorted 28C13 and 28C13-CD44 + CD117 + cells. Various stem cell markers were determined using real-time RT-PCR. ar: SD, n=3, *: P<.5, **: P<.1. G, Various amount of cells were mixed with Matrigel (1:1) and injected into nude mice subcutaneously. The formation of tumor was observed up to 3 months.

Ik mrna expression (Z-score) 2 1-1 -2-3 -3 Pearson correlation of DD2 and Ik =.169, P-Value =. -2-1 DD2 mrna expression (Z-score) 1 2 3 4 Supplementary Figure S1: The DD2 and Iκα mrna expression data in ovarian serous cystadenocarcinomas was retrieved from TCGA dataset (Nature 211) by using a tool in www.cbioportal.org. The DD2 and Iκα mrna expression Z-scores were plotted and their correlation was analyzed by using Minitab.

DD2 expression (Z-score) 4 3 2 1-1 -2-3..5.1.15.2.25.3 DD2 promoter methylation beta value (HM27) Pearson correlation of DD2 and DD2 methylation = -.9, P-Value =.849 DD2 mrna expression (Z-score) 4 3 2 1-1 -2-3 Putative copy-number calls determined using Gistic2 on the MSKCC Agilent 1M data. -2 = homozygous deletion; -1 = hemizygous deletion; = neutral / no change; 1 = gain; 2 = high level amplification. DD2 Putative CNA (GIDTIC) Supplementary Figure S11: The DD2 mrna expression data, DD2 promoter methylation data, and DD2 copy number alteration (CNA) data in ovarian cancer patients were retrieved from TCGA dataset (Nature 211) by using a tool in www.cbioportal.org. A, the relationship between DD2 mrna and promoter methylation was plotted and analyzed for their correlation by using Minitab., the effect of DD2 putative CNA on DD2 mrna expression level.

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