-8-6 -4-2 CHEK2 MKI67 RB1 RBL1 SKP2 Fold-Change Fold-Change -3-2 -1 CCND1 CCNE1 CDK4 CDK5R1 5 1 15 ATM ATR BAX BCCIP BRCA1 BRCA2 BL1AT ABL1A ABL1 CCNG1 CCNG2 CCNH CCNT1 CCNT2 CDC2 CDC34 CDKN1A CDKN1B CDKN2A CDKN2B CDKN3 CHEK1 CKS1B CKS2 CUL1 CUL2 5 1 15 CUL3 DDX11 DNM2 E2F4 GTF2H1 GTSE1 HERC5 HUS1 KNTC1 KPNA2 MNAT1 MRE11A NBN RAD1 RAD17 RAD9A RBBP8 RPA3 GADD45A SERTAD1 SUMO1 TFDP1 TFDP2 Fold-Change Fold-Change Upregulated Downregulated Figure S1
1 2 3 4 5 6 7 8 9 DLX3 Consensus P32 p21 P32 1- Consensus probe 2- Consensus probe + DLX3rp 3- Consensus probe + DLX3rp + competive sequence 4- Consensus probe + DLX3rp + unspecific competive sequence 5- Consensus probe + DLX3rp + p21 promoter specific sequence 6- p21 probe + DLX3rp 7- p21 probe + DLX3rp + competive sequence 8- p21 probe + DLX3rp + unspecific competive sequence 9- p21 probe + DLX3rp + Anti-DLX3 Figure S2
a mrna RQ WT 15 *** 1 *** 5 cko 1.5 1..5 *** *** 1.5 1..5 * *** ** 2. 1.5 1..5 Ctr Cxcl1 v-ras Ha. Ctr v-ras Ha. Dlx3 Ctr Krt1 v-ras Ha. Ctr Krt14 v-ras Ha Cytokine Concentration (pg/ml) b 2 15 1 5 ** Ad-GFP *** ** 1 8 6 4 2 Ctr v-ras Ha Ctr v-ras Ha CXCL1 Ad-DLX3 ** ** IL-1α * c Ad-GFP Ad-DLX3 mrna RQ 4 *** 1 **** 5 ** ** 2. ** ** 3 2 1 8 4 1.5 6 3 1. 4 *** 2 2 1.5. Ctr Ha Ctr v-ras Ha Ha v-ras Ctr v-ras Ctr v-ras Ha Cxcl1 Dlx3 Krt1 Krt14 Figure S3
SUPPLEMENTARY FIGURE LEGENDS Supplementary Figure 1. DLX3 affects cell cycle through a p53 dependent network. Bar graphs showing relative expression levels of cell cycle related genes in DLX3 transduced keratinocytes by RT2 Profiler PCR Array as indicated by manufacture s protocol. The results are shown as mean ± SD of three independent experiments. Red and green bars represent the up and down-regulated pathways, respectively. Supplementary Figure 2. DLX3 binding to the p21 promoter. Electrophoretic Mobility Shift Assay (EMSA) using recombinant murine DLX3 protein. Probes containing DLX3 consensus binding site and DLX3 binding site motif of the p21 promoter were used to perform the assay in presence of the recombinant protein DLX3 (DLX3rp). As a control for the competition assays, a mutated unlabeled probe was used. Supplementary Figure 3. Primary mouse keratinocytes transduced with v-ras Ha retrovirus. (a) Bar graph showing the relative expression level of Cxcl1, Dlx3, Krt1, and K14 in WT and Ras-transformed keratinocytes. (b) Bar graphs showing the cytokine concentration of CXCL1 and IL-1a from Ras-transformed keratinocytes transduced with Ad-DLX3 or Ad-GFP. Concentrations were obtained from the Bio-Plex Pro Mouse Cytokine 23-Plex Assay. (c) Bar graphs showing the relative expression level of Cxcl1, Dlx3, Krt1, and Krt14 from the same cells as the cytokine array. All results are shown as mean ± SD of three independent experiments. *P<.5. **P<.1. ***P<.1. ****P<.1.
SUPPLEMENTARY TABLES Supplementary Table 1. Pathways affected by Dlx3 Pathways Average Fold-Change p-value DNA Replication -3.417 2.18134E-38 Cell cycle -3.387 1.75887E-2 Proteasome Degradation -2.853 1.192E-12 G1 to S cell cycle control -2.321 2.2811E-27 TP53 network 2.139 4.9833E-12 Integrin-mediated cell adhesion 2.34 9.47645E-1 Alpha 6 Beta 4 signaling pathway 2.566 1.216E-15 TGF Beta Signaling Pathway 4.592 1.2632E-9 Selected by Fold-Change > ±2; p-value <.5
Supplementary Table 2. Gene expression profile in Dlx3-affected pathways: downregulated genes Ref_Seq Gene Name Fold-Change NM_7294 BRCA1-2.2 NM_1238 CCNE1-2.7 NM_57749 CCNE2-8.8 NM_1789 CDC25A -3.2 NM_1254 CDC6-3.9 NM_1134419 CDC7-3.3 NM_1798 CDK2-3.2 NM_77 CDKN2A -2.7 NM_3928 CDT1-4.4 NM_1114121 CHEK1-2.3 NM_198256 E2F6-2.7 NM_15895 GMNN -5.1 NM_216 H2AFZ -2.9 NM_18518 MCM1-6.2 NM_4526 MCM2-7.2 NM_2388 MCM3-4.7 NM_5914 MCM4-4.3 NM_6739 MCM5-5.3 NM_5915 MCM6-4.5 NM_5916 MCM7-4.1 NM_119818 ORC1-2.8 NM_1197259 ORC3-2.7 NM_14321 ORC6-2.8 NM_2592 PCNA -6.1 NM_21127 PMAIP1 (NOXA) -2.7 NM_946 PRIM1-2.3 NM_947 PRIM2-2.2 NM_947 PRIM2-2.3 NM_14814 PSMA2-2.5 NM_18955 PSMA3-3.3 NM_2815 PSMA4-2.5 NM_2816 PSMB2-2.3 NM_119137 PSMB3-2.2 NM_2788 PSMC2-2.2 NM_2794 PSMC4-2. NM_2794 PSMC6-3.1 NM_653 PSMD1-2.7 NM_2815 PSMD11-3. NM_283 PSMD11-3. NM_2816 PSMD12-5.7 NM_585 PSMD14-2.5 NM_119137 PSMD6-2.3 NM_2873 RAD17-2.7 NM_1164269 RAD51-3. NM_321 RB1-2.2 NM_2913 RFC1-2.2 NM_2915 RFC3-6.7 NM_113112 RFC5-4.6 NM_2946 RPA2-4.1 NM_2947 RPA3-2.8 NM_636 SMC1A -2.5 NM_1178138 TFDP2-2.6 NM_199858 UBB -2.2 NM_112667 UCHL1-13.9 NM_1143976 WEE1-2.4 Selected by Fold-Change < -2; p-value <. 5
Supplementary Table 3. Gene expression profile in Dlx3-affected pathways: upregulated genes Ref_Seq Gene Name Fold-Change NM_12342 BAMBI 3.1 NM_4324 BAX 3.53 NM_1198868 CAPN1 2.28 NM_13962 CAPNS1 2.46 NM_1172895 CAV1 3.1 NM_1233 CAV2 3.36 NM_61 CD44 3.5 NM_4358 CDC25B 2.76 NM_5194 CEBPB 2.99 NM_495 EIF4EBP1 2.38 NM_81 FKBP1A 2.26 NM_5252 FOS 5.42 NM_635 FST 5.46 NM_1924 GADD45A 3.1 NM_177 GSN 2.1 NM_1553 IGFBP7 2.1 NM_575 IL1A 6.79 NM_576 IL1B 8.98 NM_5544 IRS1 3.33 NM_224 ITGA3 7.95 NM_21 ITGA6 2.62 NM_213 ITGB4 2.85 NM_213 ITGB4 3.14 NM_2213 ITGB5 2.11 NM_227 LAMA3 2.7 NM_2291 LAMB1 2.8 NM_228 LAMB3 3.39 NM_5562 LAMC2 2.62 NM_2467 MYC 13.1 NM_2577 PAK2 2.1 NM_18855 PXN 2.73 NM_62 SERPINE1 15.42 NM_1134 SHC1 2.77 NM_17767 SLC39A4 3.13 NM_119821 SMAD7 2.22 NM_66 TGFB1 2.6 NM_124847 TGFBR2 4.5 NM_3244 TGIF1 3.9 NM_3246 THBS1 8.12 NM_546 TP53 3.23 NM_1114978 TP63 11.44 Selected by Fold-Change < +2; p-value <. 5
Supplementary Table 4. Differentiation-related genes in DLX3 transduced keratinocytes Ref_Seq Gene Name Fold-Change NM_436 CDH1 3.7 NM_1793 CDH3 3.9 NM_1444 FABP5 2.7 NM_6147 IRF6 2.24 NM_2213 ITGB5 2.1 NM_4235 KLF4 2.5 NM_173 KLF5 3.52 NM_6121 KRT1 2.54 NM_421 KRT1 2.1 NM_17617 NOTCH1 4.13 NM_2577 PAK2 2.1 NM_5544 IRS1 3.33 NM_224 ITGA3 7.95 NM_21 ITGA6 2.62 NM_213 ITGB4 2.85 NM_213 ITGB4 3.14
Supplementary Table 5. Biological Functions affected by DLX3 in SCC13 cells Biological Function Annotations Activation Z- score p-value Protein Synthesis 1.523 1.1E-12 Organismal Survival 1.257 1.1E-12 Cell Death and Survival.252 4.67E-14 Tissue Development -.17 4.76E-11 Cell Cycle -.378 7.35E-11 Cellular Growth and Proliferation -.899 3.87E-22 Cancer -1.542 1.2E-19 Cellular Movement -1.825 1.7E-16 Selected by Fold-Change > ±2
Supplementary Table 6: Cluster 1: Regulation of Cell Cycle genes in DLX3 infected SCC13 cells Values of Log2 fold change Ref_Seq Gene Name 18hrs 24hrs 36hrs 48hrs NM_14885 ANAPC1 -.371828991 -.874766234 -.647926778 1.521329667 NM_12247 ANAPC11 -.21913258 -.24173131.164254159 1.489211441 NM_36 AURKA.519566877 -.79818825.113457675 1.45338421 NM_14756 BEND5.6321741 -.17979784.17551 1.117752844 NM_113617 CCND3.26549964 -.85698925 1.69847716.849948153 NM_21873 CDC25B.632146 -.499945765.1934428 1.2282346 NM_113442 CDC7 -.467227265 -.53198334 -.38928124 1.89154213 NM_76 CDKN1C.174795683-2.758464818.2876896 3.19162487 NM_77 CDKN2A -.457596724 1.9668636.478974515 2.6952879 NM_78626 CDKN2C -.85487818.911862897.29369729 8.521475542 NM_14956 CEP164 -.2334361 -.9914621 -.516331386 1.31864859 NM_183314 CHMP1A -.113891542.5267527 -.39836985 1.32698148 NM_12515 CSNK1A1 -.79396717 -.5767995.234316664 1.85195365 NM_4399 DDX11.1289885 -.3234937 -.2268265 1.231949977 NM_152438 DDX11.9452622 1.3212662 -.613473538.68699382 NM_1145315 DSN1.43888.194467237.282282 1.5488561 NM_198256 E2F6 -.17449322 1.83454226 -.2254592.16518539 NR_26978 HAUS1-1.43654992.64355858.17446496 -.4578618 NM_5537 ING1 -.321724714 -.887233759 -.188431 1.14963827 NM_17383 LIN9.58828285 -.7489989.495272232 1.372581153 NM_4524 LLGL2 -.73531943 1.28434772.3221888 1.2683723 NM_355 MAD1L1 -.2681786 1.6157574 -.43852384.374487925 NM_113156 MLF1 -.561669429-1.77154551 -.11969214 1.19916586 NM_1167619 MLH1.119611812.24967833 -.1625596 1.7867912 NM_15341 NCAPH.1298765 -.3837193 -.9519229 1.23895136 NM_1135177 NEDD1 -.583531-1.268157262 -.4168636 2.1185996 NM_423 PKMYT1 -.72622812 -.28215182 -.11569428 1.5298823 NM_18344 RBL1 2.39683587-1.45387543.85222237.8789463 NM_13787 STRADA.615985896 1.256542293.35924378 1.5287354 NM_6852 TLK2.149966579 -.61199514.46853918 1.24367114 NM_181799 UBE2C.5553687-1.547219 -.126787789.25165277 NM_1451 UBE2S -.1657587.25332821.1572465 1.2451963
Supplementary Table 7: Cluster 3: Down-regulation of Apoptosis related genes in DLX3 infected SCC13 cells Values of Log2 fold change Ref_Seq Gene Name 18hrs 24hrs 36hrs 48hrs NM_1142931 API5 -.621596.23126261.1645518-1.4625223 NM_113845 BCL6 1.9929114 -.35137979.6624262 -.5361874 NM_2327 ACVR1B -.71717553 -.1568147 -.189664328-1.14316117 NM_113616 APP.926974.228868252.264482326-1.19424288 NM_6538 BCL2L11.3566253 -.756146813 -.28586192-1.16893332 NM_197967 BID -.712672833.282663969 -.527164563-1.5848112 NM_4331 BNIP3L -.19115599 -.229712 -.15213467-1.14923723 NM_1223 CASP1-1.799788734-1.548138575.81873864.75588415 NM_12114 CASP14 -.14466443.422297594.663648239-2.8411197 NM_1168389 CITED2-1.2711912 1.319164241 -.246299427-1.99239477 NM_3589 CUL4A -.454989559 -.149399972 -.13296713-2.3779795 NM_3583 DYRK2 -.4624548.9271248 -.59864355-1.2853632 NM_114376 EIF5A -.451388854.379189864 -.32794564-1.6842722 NM_1137 GSPT1 -.6569835.429475991-1.4523258-3.7559227 NM_177551 HCAR2.3445517 -.87196891 -.57774828-1.3843314 NM_1127584 ING4 2.363678599 -.32111885-1.924292189 -.521663592 NM_132282 KLF1 -.19564118 -.389231466 -.83231463-1.242454689 NM_5923 MAP3K5.14837224 -.417145994 -.5985479-1.1566713 NM_182763 MCL1 -.336553576.1416159 -.31714214-1.71311125 NM_5863 NET1 -.39356924 -.64591829 -.694795959-1.36984261 NM_257 NGFR -.79516169 -.4594732-1.21794698-1.352183271 NM_1438 NGFRAP1.137661184.231782711 1.197868272 -.2981824 NM_199185 NPM1 -.28532836 -.3726454 -.266523154-1.75593776 NM_11875 NR3C1 -.418822687 -.134672626 -.15414658-1.37322311 NM_1159999 NRG1 -.895192989 -.59385556 -.15855557-1.62365372 NM_12385 NUPR1 -.233977284.65977.5729829-1.575457388 NM_18951 PLAGL1.266734115.54846129 1.46146974 -.41186634 NM_114264 PPT1-1.761372328 -.57954918 -.29237763-1.5846436 NM_6115 PRAME.118474 -.94548235.8551233-1.3129447 NM_133582 PRKCZ -.91485 -.489838 -.1548972-1.215273586 NM_1139518 PRKRA -.749711787 -.384756642 -.768921168-2.546823137 NM_183236 RAB27A -.44472979 -.171787775.44642988-1.4465432 NM_78 RTN4 -.698985719.1129134.2927615-2.53113246 NM_1143818 SERPINB2 -.259663929.76395836 9.31289283 2.88735277 NM_114512 SMAD3 -.96554582 -.64684334.39642631-1.253794728 NM_124466 SOD2.1229965 -.22498 -.45598825-1.655722873 NM_1142299 SQSTM1.1676886 -.611225865 -.984231264-1.32714793 NM_1135599 TGFB2.26543549-1.51891.29287338 -.868987421 NM_113916 TGFBR1 -.575145992-1.352549261.815486463-1.861689551 NM_362 TIMP3 -.271316 -.534537125 -.9489448-1.543635177 NM_1145549 TXNDC5 -.4162869 -.52748858 -.6259536-1.51581262
Supplementary Table 8. qrt-pcr primer lists For human (5-3 ): Primer Name DLX3-F DLX3-R TP53-F TP53-R p21-f p21-r p57-f P57-R ΔNp63-F ΔNp63-R NOXA-F NOXA-R b-actin-f b-actin-r Primer Sequence CCCCTCGGCCAATAAGACAG GTTTGGCACCAAAACTGCCA GCCCAACAACACCAGCTCCT CCTGGGCATCCTTGAGTTCC ATGAAATTCACCCCCTTTCC CCCTAGGCTGTGCTCACTTC GCGGCGATCAAGAAGCTGTC CCGGTTGCTGCTACATGAAC GAAAACAATGCCCAGACTCAA TGCGCGTGGTCTGTGTTA GAGATGCCTGGGAAGAAGG ACGTGCACCTCCTGAGAAAA TGGATGATGATATCGCCGCGCTCG CACATAGGAATCCTTCTGACCCA For mouse (5-3 ): Primer Name Primer Sequence Dlx3-F ATTACAGCGCTCCTCAGCAT Dlx3-R CTTCCGGCTCCTCTTTCAC Tp53-F GTTCAAGGTCATCCTTCCCTG Tp53-R ACCATCGCTTTCGCACCT p21-f GTGGCCTTGTCGCTGTCTT p21-r GCGCTTGGAGTGATAGAAATCTG p57-f TAGAGGCTAACGGCCAGAGA p57-r CCCAGAGTTCTTCCATCGTC Krt1-F TCACTGAATTGAGACGTACTGTTCAGGGTCTGG Krt1-R CTTGATGTCTAGGAGTTGTTGGTACTCGGCGTT Krt14-F TCCTCTCAATTCTCTCTCAGTCATCC Krt14-R ATAGGGACAATACCTTCCAGCAGTAT ΔNp63-F GGAAAACAATGCCCAGACTC ΔNp63-R GATGGAGAGAGGGCATCAAA Cxcl1 primers were purchased from Quantitect Primers (Qiagen)