Supporting Figures

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Fig. 5. Cytoplasmic p65 distribution in colorectal cancer cells. (A) Confocal images showing the localization of p65 by immunostaining of different cell lines. Nuclear staining with PI is showing in the lower panels. Foreskin fibroblast HS27 and epithelial HEK-293 cell line are used as nontransformed controls. (B) Western blot analysis of p65 in cytoplasmic and nuclear extracts from the different cell lines. a-HDAC1 and a-tubulin are used as fractionation and loading controls. (C and D) Western blot analysis of IkBa (C) and p100/p52 (D) from the different cancer cell lines. a-Tubulin is used as a loading control.

Fig. 6. Cytoplasmic p65 and p100/p52 distribution in primary colorectal tumors. (A) Sequential sections showing IHC of p65, p52, and p50 in primary colorectal adenomas (´400). (B) ChIP with a-p52 and a-p65 antibodies from human colorectal tumor tissues CRC-X compared with normal colon tissue. PCR detection of the indicated promoters is shown. (C) Control ChIP with a-p52 antibody from untreated or LPS-treated MCF7 breast cancer cells.

Fig. 7. Specific phosphorylation of Ser-2410 of SMRT by nuclear IKK. (A) Kinase activity assay of precipitated IKKa/b tested on GST-SMRT (aa 2321-2525) and the Ser2410Ala mutant in SW480 and HS27 control cells. Phosphorylation was detected by [³²P]ATP incorporation. (B) a-P-SMRT staining in sequencial sections corresponding to biopsies shown in Fig. 1C demonstrating that IKKa activation correlates with SMRT phosphorylation in tumors. Low magnification (´200) and detailed higher magnification (´600) are shown.

Fig. 8. Phosphorylation of H3 in IKKa-target promoters in CRC-X ChIP with a-Phospho-H3 from normal tissue or patient-derived CRC-X and detection of the indicated genes by PCR.

Fig. 9. I kB a32-36 does not inhibit hes1 transcription. (A) HCT116 cells were cotransfected with hes1-luc or 3xkB-luc reporters and the indicated constructs. The graph represents the relative luciferase activity in the different conditions. The average and standard deviation from duplicates of one representative of three experiments are shown. Levels of I kB a32-36 were determined by Western blot with a-Ha antibody. (B) RT-PCR of hes1 and two different NFk B targets from HCT116 cells transfected with control plasmid or I kB a32-36.

Fig. 10. DN-IKKa restores SMRT association to the hes1 promoter leading to a decrease in gene expression. Shown is ChIP with a-SMRT and PCR detection of the hes1 promoter and RT-PCR of hes1 in HCT116 cells stably transfected with DN-IKKa or the vector alone.

Fig. 11. BAY11-7082, a specific IKK inhibitor, inhibits cell growth and promotes apoptosis in tumor cells. Shown are representative cultures of HS766 (pancreatic cancer cell line) and HT29M6 cells after 3-day treatment with DMSO or BAY11-7082. (Left) Graphs represent daily cell counts of the indicated cell lines incubated at 5 mM and 10 mM of BAY11-7082. (Center) Photographs show a representative image of the cultures at day 3 of 10 mM treatment. Images were obtained in an Olympus IX-10 at ´100. (Right) Flow cytometry analysis of AnnexinV staining from a representative 2-day culture at the indicated dose.

Fig. 12. Sequence of primers used for RT-PCR and PCR for ChIP analysis.