Supplementary Table S1. Primary antibodies for Western blot analysis. Antibody name and specifications
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1 Supplementary Table S1. Primary antibodies for Western blot analysis. Antibody name and specifications Manufacturer Actin (C-11) sc-1615, Santa Cruz, CA Akt1 (B-1) sc-5298 GAPDH (6C5) sc JAK2 (HR-758) sc-278 PDGFR-α (C-20) sc-338 phospho-pdgfr-α (Tyr 720) sc r Ron β (C-20) sc-322 p-ron β (Tyr1238/ Tyr1239) sc Raf-1 (E-10) sc-7267 Stat3 (F-2) sc-8019 Stat5 (C-17) sc-835 Stat6 (STAAD23A) sc Trk (C-14) sc-11 Phospho-MSPR/RON (Y1238/Y1239) AF1947 R&D Systems, Minneapolis, MN Caspase-3 #9662 Technology, Danvers, MA p44/42 MAPK (Erk1/2) (3A7) #9107 Phospho-Akt (Ser473) (D9E) #4060 Phospho-c-Raf (Ser338) (56A6) #9427 Phospho-Jak2 (Tyr1007/1008) #3771 Phoshpo-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D E) #4370 Phospho-Stat3 (Tyr705) #9131 Phospho-Stat5 (Tyr694) (C11C5) #9359 Phospho-TrkA (Tyr490) #9141 Phospho-Stat6 (Y641) # BD Transduction Laboratories, San Jose, CA
2 Supplementary Table S2. Primer sequences for qrt-pcr. Primer name Primer sequence GADD153-for 5 -TGAGCGTATCATGTTAAAGATGAGCG-3 GADD153-rev 5 -GGTGTGGTGATGTATGAAGATACACTTCC-3 GADD34-for 5 -TGATCCGGACCCTGAGACTCC-3 GADD34-rev 5 -CCCAGACAGCCAGGAAATGG-3 LIPH-for 5 -TCTGGGTTTGTTGGAGAGATGTACG-3 LIPH-rev 5 -CTGGGATCTAATCTGTCTTGGTGAGG-3 PIM2-for 5 -CAAAGTGATTCCCCGGAATCG-3 PIM2rev 5 -ACCCACTTTCCATAGCAGTGCG-3 PPIA-for 5 -TGGACCCAACACAAATGGTTCC-3 PPIA-rev 5 -CATGGCCTCCACAATATTCATGC-3 SOS2-for 5 -CGAGGAGAACAGTCCGAAATGG-3 SOS2-rev 5 -AGAGAGACTCTTCATTAGCTGAGAGAGTGG-3 STAT4-for 5 -TGGCTGCAGCCAACATGC-3 STAT4-rev 5 -GCCACTTTGTGCTCCACATTCC-3 TNFSF10-for 5 -CAGTCTCTCTGTGTGGCTGTAACTTACG-3 TNFSF10-rev1 5 -CATCTTCTTTTAAGAAACAAGCAATGCC-3 TNFSF10-rev2 5 -AGCAATGACCCTGTCTGCTGC-3 TNFSF8-for 5 -TCTTCACGGTGGCCACTATTATGG-3 TNFSF8-rev 5 -TTCTGAGCAATTTCCTCCTTTGAGG-3 TRAF3IP3-for 5 -GACCTACTGTCCACTCTGATTCAGGC-3 TRAF3IP3-rev 5 -GGGTACAGGTGTATAGCTGGTTTTCTAAGG-3
3 Supplementary Table S3. TKIs and conventional chemotherapeutics used in this study. Compound Solvent Dasatinib (LC Laboratories, Woburn, MA) Doxorubicin (Merck Chemicals, Darmstadt, Germany) Erlotinib (LC Laboratories) Gefitinib (LC Laboratories) Lapatinib (LC Laboratories) Lestaurtinib (LC Laboratories) Sorafenib (LC Laboratories) Sunitinib (LC Laboratories) Vinblastine (Sigma-Aldrich, St. Louis, MO) Vandetanib (LC Laboratories) Vatalanib (LC Laboratories) H 2 O TKIs and conventional chemotherapeutics used in this study and their solvents. Supplementary Table S4. Sensitivity of Hodgkin-Reed/Sternberg cell lines to tyrosine kinase inhibitors (TKIs) at concentrations achievable in patients. % of viable cells TKI L-1236 L-428 KM-H2 Dasatinib 1 µm 48 h Lapatinib 3 µm 72 h Sunitinib 300 nm 72h Percent of viable cells after treatment with TKIs dasatinib, lapatinib and sunitinib at concentrations achievable in patients. TKIs showing inhibitory effects on HRS cell lines in the initial screening at concentrations of 5 µm were applied at concentrations achievable in the plasma of patients and compared to a solvent treated control at optimal growth conditions. At the indicated achievable concentrations the TKIs did reduce the number of viable cells below 80%.
4 Supplementary Table S5. Amount of phosphorylated RTK in relation to total RTK amount. Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6 Figure 3A p-trk in L p-trk in HDLM Figure 3B p-ron in L Figure 3C p-pdgfrα in U-HO Figure 3D p-pdgfrα in KM-H Figure 3E p-pdgfrα in KM-H Densitometric analysis of Western blots shown in Figure 3. The relative phosphorylation of RTKs was normalized to the detected total amount of RTK and then compared to the solvent treated control sample (lane 1) of each experiment.
5 Supplementary Table S6. Genes with increased expression upon lestaurtinib treatment of KM-H2 as determined by GeneChip Human 1.0 ST Arrays. Absolute fold increase P-value Gene symbol Absolute fold increase P-value Gene symbol 3, , TCP11L2 2, , NCF2 3, , GPR155 2, ,86E-04 SOS2 3, , DDIT4 2, , GPR15 2, , PIK3IP1 2, , CES3 2, , EVI2B 2, , IFIT2 2, ,84E-04 SH3D19 2, , LPXN 2, , TNFSF8 2, , DEPDC6 2, , ABAT 2, , C3AR1 2, , ANTXR2 2, , TRAF3IP3 2, , KLHL24 2, , SLFN5 2, , CECR1 2, , RGS1 2, , F2RL2 2, , HIST1H2BK 2, , CAP2 2, , C3orf59 2, , TMEM156 2, , TMEM229B C14orf83 2, , LIPH 2, , SLC2A3 2, , ABHD4 2, ,48E-04 CYTH1 2, , YPEL3 2, , FBXO32 2, , NEDD9 2, , TLR6 2, ,60E-04 C4orf34 2, , ACSF2 2, ,62E-04 ACTA2 2, , STAT4 2, , YPEL2 2, , RARRES3 2, , PIM2 2, , TRGV3 TARP 2, , GOLGA5 2, , FCGBP 2, , ENPP2 2, ,15E-04 PDGFRA 2, , CCDC92 2, , CD82 2, , TFEC 2, , CD48 2, , TFPI 2, , ENTPD1 2, , VWA5A 2, , DAP 2, , BTG2 2, , LOC , ,94E-04 TNFSF10 2, , CASP9 2, , MVP 2, , LY75 CD302 2, , ST6GAL1 Absolute fold increases of genes in KM-H2 cells treated with 1 µm lestaurtinib for 24 h compared to a solvent treated control calculated from duplicate DNA microarray analyses by pairwise comparison (p < 0.05). Listed are 63 genes with fold changes > 2.
6 Supplementary Table S7. Genes with decreased expression upon lestaurtinib treatment of KM-H2 as determined by GeneChip Human 1.0 ST Arrays. Absolute fold P-value Gene symbol Absolute fold P-value Gene symbol decrease decrease 4, , HIST1H1B 2, , KIF2C 3, , KIF20A CDC23 2, ,69E-04 LOC , , PBK 2, ,35E-04 KIFC1 3, , HIST1H2AB 2, , CKS1B LOC , , FOXM1 2, ,84E-04 CDC20 3, , MYBL2 2, , H2AFX 3, , CDK1 CDC2 2, , HIST1H1A 3, ,52E-05 ANLN 2, , E2F8 3, , NCAPG LCORL 2, ,61E-04 CENPN C16orf61 3, , NUF2 2, , CENPA 3, , CDCA2 2, , UBE2C 3, , ESPL1 2, ,77E-04 GMNN 3, , CCNA2 2, , CENPK 3, , ASPM 2, , HIST2H2AB 3, , UHRF1 2, , NUSAP1 3, ,00E-04 CKAP2L 2, , CSNK1G1 LOC , , FAM72D FAM7 2, , SKA1 2A FAM72B FA M72C 3, , SPC25 2, , ERCC6L 3, , FAM72D FAM7 2, , WEE1 2A FAM72B FA M72C 3, , KIF23 2, , CCNF 3, , FAM72D FAM7 2, , PEG10 2A FAM72B FA M72C 3, , RRM2 2, ,83E-05 PRC1 3, ,58E-05 SPAG5 SGK494 2, ,20E-04 Transcripts Cluster Id: , , PLK1 2, , AURKA 3, , HIST1H2BM 2, , DHFR 2, , FAM72D FAM7 2, , TK1 2A FAM72B FA M72C 2, , CASC5 2, , CENPW C6orf1 73 2, , KIF15 2, ,70E-04 SGOL2 2, , AURKB 2, , UBE2S 2, , DLGAP5 2, , CDCA7L 2, , CENPE 2, , FAM54A 2, , ARHGAP11A 2, , E2F7 2, , KIF4A 2, , HIST1H4B 2, , DHRS2 2, , KIAA0101 CSN K1G1 2, ,57E-04 MCM10 2, , MND1 2, , NDC80 2, , Transcripts Cluster Id: , , BUB1B PAK6 2, , CIT 2, , CDCA3 2, , ZWINT 2, , KIF18B 2, , STIL 2, ,38E-04 ESCO2 2, , Transcripts Cluster Id: , , FAM111B 2, , DHFR 2, , DLEU2 2, , XRCC2 2, , CDCA8 2, , PIGW 2, , PLK4 2, , CDC45 CDC45L 2, , GINS1 2, ,78E-04 RAD51 2, , KIFC1 2, , MAD2L1 2, , KIF14 2, , NCAPH 2, , HJURP 2, , KIF20B 2, , BUB1 2, , CCNB2 2, , EXO1 2, , TUBB2C 2, , CDKN3 2, , WEE1 2, , CDCA5 2, , KIF18A
7 2, , KIAA1524 2, , RFC3 2, , HIST2H3A HIST 2, , DSCC1 2H3C HIST2H3 D 2, , HIST2H3A HIST 2, , RAD54L 2H3C HIST2H3 D 2, ,61E-04 HIST1H2BI 2, ,57E-04 SPC24 2, , SLC29A1 2, , TMPO 2, ,08E-04 KIF18B 2, , MRTO4 2, ,61E-04 BIRC5 2, , FAM83D 2, ,54E-04 HMMR 2, , TRAIP 2, , SKA3 MRP63 2, ,93E-04 PRR11 2, , RAD51AP1 2, , C11orf82 2, , DEPDC1 2, ,06E-04 C3orf26 2, , CENPF 2, , PKMYT1 2, ,16E-04 OIP5 2, , Transcripts Cluster Id: , , TPX2 2, , PTTG1 2, , HIST2H3D HIST 2, , TROAP 2H3A HIST2H3 C 2, , CCNB1 2,082 0, GTSE1 2, , TTK 2, , HIST1H3D HIST 1H2AD 2, , HIST1H4A 2, , RACGAP1 2, , NEK2 2, , GSG2 2, , HMGB2 2, , CKS1B 2, , CENPI 2, , NEIL3 2, ,40E-04 TACC3 2, , CENPH 2, , CEP55 2, , PRIM1 2, , HIST2H3D HIST 2, , ECT2 2H3A HIST2H3 C 2, , C15orf42 2, , HIST1H2BF 2, , HIST1H2AK HIS 2, , HIST1H3G T1H2BN 2, , CKS1B LOC , ,47E-04 PCNA PCNAAS 2, , CDC6 2, , TRIP13 BRD9 2, , UBE2T 2, , NET1 2, , TOP2A 2, ,71E-05 ARHGAP19 2, , ORC1L 2, , MMP12 2, , HIST1H2AH 2, , ANP32E 2, , DIAPH3 2, , FARSB 2, , KIF11 2, , DTL 2, , MKI67 2, , CCDC99 2, ,04E-04 MELK 2, , BRCA2 2, , SGOL1 2, , ATAD2 2, , UBE2S 2, ,83E-04 DNA2 Absolute fold decreases of genes in KM-H2 cells treated with 1 µm lestaurtinib for 24 h compared to a solvent treated control calculated from duplicate DNA microarray analyses by pairwise comparison (p < 0.05). Listed are 180 genes with fold changes > 2.
8 Supplementary Table S8. Gene ontology annotation of differentially expressed genes in lestaurtinib treated KM-H2 cells. Number of annotated genes Gene ontology annotation Description (biological process) Decreased expression 56 GO: mitotic cell cycle 52 GO: cell division 47 GO: cell cycle 39 GO: mitosis 32 GO: M phase of mitotic cell cycle 29 GO: mitotic prometaphase 20 GO: nucleosome assembly 18 GO: cell proliferation 16 GO: chromosome segregation 15 GO: DNA replication Increased Expression 8 GO: signal transduction 7 GO: immune response 5 GO: apoptosis 4 GO: G-protein coupled receptor protein signaling pathway 4 GO: lipid catabolic process 3 GO: response to virus 3 GO: positive regulation of IκB kinase/nf-κb cascade 3 GO: activation of caspase activity 3 GO: cell proliferation Gene ontology (GO) analyses of genes with decreased or increased expression (fold changes > 2) after 24 h treatment of KM-H2 cells with 1 µm lestaurtinib compared to a solvent treated control determined by GeneChip Human 1.0 ST Array. Of 180 downregulated genes 116 were associated with the ten most strongly represented GO annotations shown here. Of 63 upregulated genes 25 are represented in the nine GO annotations listed.
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TFDP2 TFDP1 SUMO1 SERTAD1 RPA3 RBBP8 RAD9A RAD17 RAD1 NBN MRE11A MNAT1 KPNA2 KNTC1 HUS1 HERC5 GTSE1 GTF2H1 GADD45A E2F4 DNM2 DDX11 CUL3 SKP2 RBL1 RB1
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