Supplementary Data

Supplementary Figure 1. Sabates-Bellver et al. * Supplementary Figure 1. Unsupervised hierarchical clustering of normal colonic mucosa (32 samples, green branches), colorectal adenomas (32 samples, red branches), and advanced colorectal carcinomas (25 samples, black branches) based on transcriptome analysis with Affymetrix U133 Plus 2.0 arrays. Data were analyzed as described in Methods. The 32,813 probes plotted on the y-axis (i.e., those expressed in at least one of the three tissue groups) are color-coded to indicate their expression levels relative to the median level for the gene across the entire sample set (blue: lower; red: higher). As expected, the tissue sample collected from patient NM’s polyp (*) clustered with the adenomas. This sample was clearly adenomatous although the polyp itself was classified as a T1 tumor (Table 1). Supplementary Figure 2; Sabates-Bellver et al. A B mitosis humoral defense mechanism CDC25B CCR2 CDC2 CCL14 CCNF C7 RAN CCL14 CDCA5 TNFRSF17 MAD2L1 CLU MAD2L1 CR2 CCNB1 C1S PRMT5 TNFRSF7 BUB1B SERPING1 PBK CD53 TPX2 KLRK1 BUB1 LY9 CDC2 CSF1R MAD2L2 KLRC4 CDC20 CLEC2B CDC6 C1QC UBE2C C1QA NUSAP1 C1QB AURKA HHEX BUB1 C1S CDC2 KLRC1 CCNA2 KLRD1 HCAP-G ITGB2 PTTG1 SPINK5 SMC2L1 HHEX PLK1 CD163 CCNA2 CCR2 KNTC2 MAG NEK2 CD163 KIF2C C3 BRRN1 XCL1 CCNB2 XCL2 NOLC1 ITGB2 KIF23 KLRB1 HCAP-G SLAMF1 KIF2C KLRD1 RCC1 LY9 AURKA CLEC2D CCNB1 CD1C CDC25C CD53 KIF11 CR1 ASPM CD74 CDC6 IGSF2 NEK2 CLEC2D KIFC1 CLU CDC25C CLU CENPE CCL18 NUSAP1 KLRF1 CDC25A CCL18 CCNF CR1 CDC25A CD40 CDC2 CCR7 adenomas normals CCR6 adenomas normals C D inflammatory response inflammatory response IL8 CCR2 ALOX5 NR3C1 IL8 NR3C1 MIF NR3C1 CXCL11 CXCL12 CXCL3 NR3C1 RIPK2 NR3C1 TLR4 AOC3 CXCL1 CD14 CXCL11 TLR7 CXCL2 STAB1 PF4 CXCL13 TLR4 CCL23 TLR4 HDAC9 CCL20 CCL21 TLR4 CCL5 CCL24 CCL5 S100A8 LY86 ALOX5 ITGAL S100A9 CCL23 REG3A CCL8 ANXA1 STAB1 SERPINA3 CCL5 IL1B CXCL12 IL1B CCL19 CCL26 BDKRB1 adenomas normals IL1R1 SIGLEC1 ADORA3 CCL2 PREX1 TLR3 Supplementary Figure 2. Tile plots TLR8 showing differential expression in C3AR1 adenomas (vs. normal mucosa) of ITGB2 genes involved in mitosis, humoral PREX1 defense, or inflammatory response. AIF1 Mitosis (A) was the biological process CCR2 category most significantly over- TLR7 represented among genes that were TLR10 overexpressed in adenomas; humoral CCL13 defense mechanisms (B) predominated C3 among those that were under-expressed ITGB2 in adenomas. The category AIF1 inflammatory response was AIF1 overrepresented in both gene lists KNG1 (over- and under-expressed in LY96 adenomas) (panels C and D). Red tiles: CCL18 high expression; green tiles: low TLR10 expression. For genes listed in gray, CCL18 two or more probes were differentially CD40 CCR7 expressed. adenomas normals Supplementary Figure 3, Sabates-Bellver et al. A 2.5 y t i s 2.0 n e t n 1.5 I d e z 1.0 i l a m r 0.5 o N 0.0 Normal Mucosa Adenomas Samples B Ct KIAA1199_Co >31 22 >31 KIAA1199 Ad 20 KIAA1199_NM 18 KIAA1199_NM >31 GAPDH NM & Ad 16 25.3 e KIAA1199_Ad c n 14 KIAA1199_Ad 24.8 e c 12 s GAPDH_Co >31 e r 10 o GAPDH_NM 20.5 u l 8 F GAPDH_NM 20.9 6 KIAA1199 NM 4 GAPDH_Ad 20.5 2 GAPDH_Ad 19.6 Co 0 -2 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 Cycles of amplification C KIAA1199 GAPDH M Co NM Ad NM Ad M Co NM Ad NM Ad 200 bp Supplementary Figure 3. KIAA1199 messenger RNA expression in colorectal adenomas. (A) KIAA1199 mRNA levels detected in samples of normal colorectal mucosa and adenomas with the Affymetrix U133 Plus 2.0 array based on normalized raw signal values (y-axis). Levels in adenomas were 54.8 times higher than those found in the normal mucosa. This difference was highly significant (p < 0.0001; bars: confidence intervals). (B) Real-time RT-PCR amplification of the coding region of KIAA1199 and the control housekeeping gene GAPDH in two representative adenomas and matched samples of normal mucosa. (A total of 15 tissue pairs were examined in triplicate, including five collected after the present study was completed.) Ct (threshold cycle): number of cycles (x-axis) at the beginning of the log phase of amplification. After 35 cycles of PCR, KIAA1199 was amplified in adenomas but not in the corresponding normal mucosa. At 40 cycles, a PCR product was also detected in many samples of normal mucosa (data not shown). (C) The KIAA1199 amplification products were subjected to agarose-gel electrophoresis to verify the specificity of the amplification. M: 50-bp ladder marker; Co: negative control; NM: normal mucosa; Ad: adenoma. Sabates-Bellver et al.

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