Fig 2: CREB expression in CRC by IHCImmunostaining confirmed the presence of CREB expression in CRC (A). Compared to normal, the expression of CREB was markedly increased in primary CRC lesion or metastatic CRC lesions (A). (B) IHC confirmed the expression status of CD31, CREB, and p53 in CRC (B). Compared to R72 CRC, the expression of CD31 (tumor angiogenesis) or CREB was markedly increased in P72 CRC. IHC analysis revealed that tumors were found to be negative for p53 expression (p53 wild type) (B).

Fig 3: P72 phenotype of p53 is associated with abundance of tumor promoting phenotypesThe expression of p-NF-kB, p-STAT, CXCR4 and p-ERK was found to be higher in the cell model of P72wt or mutant phenotypes than in the vector-control or R72wt cell models.

Fig 4: MCF-10A and HCT116 cells with stable silencing of CEACAM1 exhibit normal regulation of p21/pRb in response to DNA damage. (a) MCF-10A cells stably transfected with CEACAM1 shRNA vector no. 25 (CEACAM1-KD 025) or with a control shRNA vector (CTRL) were incubated in the presence or the absence of 1.47 nM NCS for the indicated time points, lysed, and analyzed for p21/Waf1, pRb Ser 807/811-p, pRb, or ß-actin by western blotting. (b) MCF-10A cells stably transfected with CEACAM1 shRNA vector no. 25 (CEACAM1-KD 025) or with a control shRNA vector (CTRL) were incubated in the presence or the absence of NCS 1.47 nM for 24 h in duplicate as indicated, lysed, and analyzed for p21/Waf1, pSer15-p, p53, or ß-actin by western blotting. (c) HCT116 cells stably transfected with CEACAM1 shRNA vector no. 26 (CEACAM1-KD 026), with CEACAM1 shRNA vector no. 28 (CEACAM1-KD 028) or with a control shRNA vector (CTRL) were incubated in the presence of 20 µM etoposide or dimethyl sulfoxide (DMSO (solvent)) for 1 h, washed with phosphate-buffered saline and incubated in new medium for 3 days. At the end of the incubation the cells were lysed and analyzed for p21/Waf1, pRb Ser 807/811-p or pRb by western blotting.

Fig 5: Induction of CEACAM1 by NCS or X-Rays in MCF-10A, HCT116 or SW48 cells is dependent on p53. (a) MCF-10A cells were transfected with p53 siRNA no.3 or a control (CTRL) siRNA as indicated. Two days later, 10 µM KU-55933 (K) or dimethyl sulphoxide (DMSO) (D) (solvent) was added. One hour later the cells were incubated in the presence or the absence of 20 nM NCS as indicated for an additional 4 h. At the end of the incubation total RNAs were purified and analyzed for the levels of the indicated genes by quantitative real-time PCR in triplicates. Error bars indicate s.d. within the internal replicates. (b) HCT116 or SW48 cells with wild type or inactivated p53 were irradiated with the indicated doses of X-rays and incubated at 37 °C for 6 h. At the end of the incubation total RNAs were purified and analyzed for the levels of the indicated genes by quantitative real-time PCR in triplicates. Error bars indicate s.d. within the internal replicates. (c) MCF-10A cells were incubated for the indicated times in the presence of 10 µM nutlin-3, or for 6 h in the presence of DMSO (D) (solvent) (left) or for 6 h in the presence of the indicated concentrations of nutlin-3 or the same volume of DMSO (D) (right). At the end of the incubation total RNAs were purified and analyzed for the levels of the indicated genes by quantitative real-time PCR in triplicates. Error bars indicate s.d. within the internal replicates. (d) HCT116 cells with wild type or inactivated p53 were incubated in the presence or the absence of NCS 5.46 nM for 16 h in duplicate as indicated. At the end of the incubation the cells were lysed and analysed for the levels of CEACAM1 or NBS1 by western blotting.