RARα2 expression confers myeloma stem cell features. Ye Yang, Jumei Shi, Giulia Tolomelli, Hongwei Xu, Jiliang Xia, He Wang, Wen Zhou, Yi Zhou,

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1 RARα2 expression confers myeloma stem cell features Ye Yang, Jumei Shi, Giulia Tolomelli, Hongwei Xu, Jiliang Xia, He Wang, Wen Zhou, Yi Zhou, Satyabrata Das, Zhimin Gu, Dana Levasseur, Fenghuang Zhan, and Guido Tricot Supplemental Data Table S1, related to Figure 1. Supplemental Experimental Procedures

2 Supplemental Data Table S1: Differentially Expressed Genes between CD138 + with CD138 - MM Cells Probe Set Name Gene Symbol Chromosomal Location CD138 + CD138 - p ttest CD138 - /CD _x_at _x_at _at RARA chr17q _at _at SH3GL3 chr15q _at _at _at _at LOC /// SMA4 chr5q _x_at E _at LOC chr10q _x_at LOC chr20p _at SCD5 chr4q _x_at _x_at TRIM4 chr7q22-q _x_at _at _at _x_at NLN chr5q _x_at _x_at _x_at _s_at ANKRD20A1 /// chr2q11.1 /// _x_at LOC /// LOC chr3p _at _at LOC /// WBSCR19 chr7p13 /// chr7q _x_at ZNF160 chr19q _x_at _x_at SLC25A16 chr10q _x_at _x_at SLC35E1 chr19p _at _x_at ERN2 chr16p _at FAM91A2 chr1q _x_at _x_at RANBP2 /// chr2p11.2 /// _x_at _x_at OPHN1 chrxq _x_at FLJ45803 chr11q

3 206169_x_at ZC3H7B chr22q _x_at _x_at _x_at PRDX2 chr19p _x_at RECK chr9p13-p _x_at _x_at ALMS1 chr2p _at PMS2L5 chr7q11-q _x_at DLGAP4 chr20q _x_at _x_at _x_at _x_at _x_at PRO _x_at _x_at _x_at FKSG49 chr13q _x_at PGF chr14q24-q _x_at DDX59 chr1q _s_at PDCD6 chr5pter-p _x_at DDX58 chr9p _x_at FBXW12 chr3p _x_at _at _x_at SEPP1 chr5q _x_at _x_at ADH4 chr4q21-q24 4q _x_at SPG21 chr15q21-q _at GUSBP1 chr5q _x_at _x_at IL17RA chr22q _x_at UACA chr15q22-q _at LOC chr10q _x_at _x_at DNAH3 chr16p _x_at USP34 chr2p _x_at LOC91548 chr1q _x_at C10orf104 chr10q _x_at _x_at _x_at _x_at HCG2P7 chr6p _x_at C18orf45 chr18q _x_at PDE4C chr19p _x_at RIOK3 chr18q _at JOSD3 chr11q _at RPS24 chr10q22-q _x_at

4 234788_x_at _at LOC chr12q _x_at OCIAD1 chr4p _x_at ATP8B1 chr18q21- q22 18q _at SNHG7 chr9q _x_at _at NBPF10 /// NBPF11 chr1q _at HIST2H4A /// HIST2H4B chr1q _x_at _x_at SFRS2IP chr12q _x_at CDC2L5 chr7p _s_at RBBP6 chr16p _x_at LOC chr19q _x_at _x_at NDUFS8 chr11q _at _x_at NACAP1 chr8q _x_at _x_at ATF7IP chr12p _x_at LOC chr1q _x_at SPN chr16p _x_at _x_at LOC chr4p _x_at _at TRA2A chr7p _x_at _at SYMPK chr19q _at POLR2J4 chr7p _x_at _at _x_at _at WDR42A chr1q22-q _x_at POLR1B chr2q _x_at FAM128B chr2q _x_at MPV17L chr16p _at FAM39DP chr15q _x_at DIP2A chr21q _x_at _x_at _x_at CMBL chr5p _x_at _s_at LOC chr7p _at SMEK2 chr2p _x_at _x_at _at DKFZp547E087 chr18p

5 225899_x_at FLJ45445 chr11p _at E _x_at LOC chr7q _at LOC chr16p _x_at NUMBL chr19q13.13-q _at ANKRD10 chr13q _x_at DBT chr1p _at POLR2J4 chr7p _x_at MRP _at EIF3B chr7p _x_at SAPS2 chr22q _x_at KIAA0894 chr10q _at PLA2G4B chr15q11.2-q _x_at CXYorf1 chr15q _at ZNF548 chr19q _s_at ZNF207 chr17q _at _at _at ANKRD11 chr16q _at _x_at _at _x_at JRK chr8q _x_at _s_at CSNK1A1 chr5q _at MLL4 chr19q _at HNRPA3 chr2q _x_at LOC chr1p E _x_at _x_at LOC chr16p13-p _at SCARNA15 chr15q _s_at ABCA7 chr19p _x_at LOC chr13q _s_at _at GAK chr4p _x_at FAM39B chr2q _at FLJ10404 chr5q _at SFRS4 chr1p _x_at LOC chr16p _at CC2D1A chr19p _s_at SRRM2 chr16p _at KRIT1 chr7q21-q _s_at ARHGEF1 chr19q _at _at SOLH chr16p _x_at LOC chr9q _at GATAD2B chr1q _at BTBD14B chr19p E

6 218155_x_at TSR1 chr17p _s_at SUPT5H chr19q _x_at _s_at AKAP8L chr19p _at ANKRD46 chr8q _x_at DKFZP686M019 9 chr5q12.2-q _at CCDC131 chr12q _at ATP5E chr20q _x_at CDC2L1 chr1p _at STK11 chr19p _s_at LOC chr17q _s_at GGA3 chr17q _at TMEM29 chrxp _at GBF1 chr10q _at ZRANB1 chr10q _s_at CLK4 chr5q _at E4F1 chr16p _at C6orf89 chr6p _s_at ARFGAP1 chr20q _at ZNF142 chr2q34-q _at AGPAT1 chr6p _s_at SFRS8 chr12q _at VPS13B chr8q _at ZBTB7A chr19p _at _s_at RELA chr11q _s_at SF4 chr19p _at DHX38 chr16q21-q _at AAMP chr2q _s_at CLINT1 chr5q23.1-q _s_at SAMM50 chr22q _x_at GANAB chr11q _at C16orf63 chr16p _at CNPY3 chr6pter-p _at RTTN chr18q _at C14orf94 chr14q _at SACM1L chr3p _s_at CAND1 chr12q _at LUC7L2 chr7q _x_at NONO chrxq _at PITPNB chr22q _s_at PCMT1 chr6q24-q _at MORF4L2 chrxq _x_at SIP1 chr14q _at TMEM14A chr6p _s_at DIABLO chr12q _at SMC1A chrxp11.22-p

7 202395_at NSF chr17q _s_at ABHD10 chr3q _at HN1 chr17q _at CEP152 chr15q _s_at KIF18A chr11p _x_at IFNAR2 chr21q q _at POLQ chr3q _s_at KIAA0101 chr15q _x_at MC1R /// TUBB3 chr16q _x_at FBXO5 chr6q25-q _s_at GINS3 chr16q _x_at TUBA1C chr12q12-q _at C12orf32 chr12p _x_at TUBA1B chr12q _s_at BUB3 chr10q _at SLC30A1 chr1q32-q _at SGOL2 chr2q _at TK1 chr17q23.2-q _at TUBG1 chr17q _at FAM111A chr11q _s_at CKS1B chr1q _at CKAP2L chr2q _at TACC3 chr4p _s_at RAD51 chr15q _at WEE1 chr11p15.3-p _s_at TSPAN3 chr15q _at CCNB1 chr5q _at KIF11 chr10q _at HYLS1 chr11q _x_at TUBB chr6p _s_at RACGAP1 chr12q _s_at THRAP3 chr1p _at FANCI chr15q _s_at KIAA1333 chr14q _at CDCA5 chr11q _s_at TPX2 chr20q _at NDC80 chr18p _at NUSAP1 chr15q _s_at RFC5 chr12q24.2-q _at ARL6IP1 chr16p12-p _a_at MAD2L1 chr4q _s_at ANLN chr7p15-p _at FAM54A chr6q _at DLG7 chr14q _x_at TUBB2C chr9q _at RRM2 chr2p25-p _at HMMR chr5q33.2-qter

8 218755_at KIF20A chr5q _at CCNF chr16p _s_at KIF2C chr1p _s_at KPNA2 chr17q _at CCNA2 chr4q25-q _s_at PRC1 chr15q _x_at C18orf24 chr18q _at E2F8 chr11p _at HJURP chr2q _s_at KIFC1 chr6p _at MELK chr9p _s_at AURKA chr20q13.2-q _s_at TOP2A chr17q21-q _s_at KIF23 chr15q _at FAM83D chr20q11.22-q _x_at CDC2 chr10q

9 Supplemental Experimental Procedures Microarray data analysis Plasma cell purifications and gene expression profiling, using the Affymetrix HuGene-1_0-st-v1 microarray, were performed as previously described by Zhan et al Signal intensities were preprocessed and normalized by GCOS1.1 software (Affymetrix). A total of 16 myeloma cell samples (CD138 + and CD138 - ) from 8 myeloma cell lines were used in this study. The Scaling Factor (SF) of each chip was normalized to 500. Affymetrix signals were transformed by the logbase 2 for each sample. The genes defined as drug-resistant related in the analysis were chosen as follows. We compared gene expression profiles from 8 paired myeloma cell lines using a paired t test. A total of 291 probe sets were significantly differentially expressed with p < 0.01 and the average Signal in the higher expression should be > 500) (Supplemental Table 1). Supervised cluster analysis of known classes was performed for Figure 1B using GeneCluster2 (Broad Institute, Cambridge, MA). Cell culture and Sorting ARK, KMS11, ARP1, OCI-MY5 and 5TGM1 et al. cell lines preserved in our lab were cultured in RPMI 1640 containing 2mM L-glutamine, 10% heat-inactivated fetal bovine serum (FBS), penicillin (100U/mL), and streptomycin (100 μg/ml) in a humidified incubator at 37 C in 5% CO2. Cultures were established by initial suspension at 4X10 5 viable cells/ml and maintained at cell concentrations between 5X10 5 and 1X10 6 viable cells/ml. Medium was changed 2 to 3 times per week. We used automacs and CD138 microbeads (Miltenyi Biotec Inc., Auburn, CA) for sorting since automacs system has lower pressure and shorter time period than flow cytometry sorter. To avoid non-viable cells after sorting by our experience, following points are critical. A) Total amount of cells should be more than 20M with viability above 95% before sorting. B) CD138-

10 microbeads incubation for only 20mins at 4 degree Celsius, gently shake incubation tube every 5 minutes. C) Using depletion protocol for automacs separator. D) Add 5ml of RPMI 1640 with 10% FBS and 1% PSG to collecting tube for CD138 +/- cells. After sorting, cell viability was checked again by trypan blue to confirm that the viability was around 90% for the following cell growth and Real time PCR study.