Figure 1.
Endogenous expression of S100A6 in the MKN45 gastric cancer cell line.
(A) Endogenous expression of S100A6 in MKN45 gastric cancer cells was detected by western blot. (B) Immunofluorescent staining of S100A6 in MKN45 cells. (a) Immunofluorescent staining of S100A6 with anti-S100A6 antibody (in green); (b) Detection of nuclei with propidium iodide (in red); (c) Merged picture.
Figure 2.
Interaction of S100A6 and CacyBP/SIP in the MKN45 gastric cancer cell line.
(A) Western blot analysis of the transfectants CacyBP/SIP and CacyBP/SIPΔS100 transfectants with anti-FLAG and anti-CacyBP antibodies. β-actin was used as a loading control. (B) Co-immunoprecipitation assay to detect an interaction between S100A6 and CacyBP/SIP in MKN45 cells. After incubation with anti-CabyBP antibody, the binding sites were detected by anti-S100A6 antibody.
Figure 3.
Effects of CacyBP/SIP on cell proliferation in vitro.
(A) Detection of the cell growth rate in vitro. Cell number was evaluated by absorbance at 490 nm using the MTT assay at the indicated times. The value shown is the mean of three determinations. (B) Cell cycle distribution and proliferative indices (PI) of the transfected cells. The cells were detergent extracted, stained with propidium iodide, and analyzed by flow cytometry. PI = (G2+S)/(G1+S+G2).
Figure 4.
Effects of CacyBP/SIP transfectants on tumorigenesis.
(A) Detection of the clonogenic formation in medium. (B) Detection of the clonogenic formation in soft agar. Cells were placed in media containing soft agar or on a plate and incubated for 20 days. The number of foci >100 µm was counted. Vertical bars represent the mean ± SD from at least three separate experiments, each conducted in triplicate. (C) In vivo tumorigenicity was evaluated by a nude mice assay. Mice were injected subcutaneously with 1×107 transfected cells. The volume of each tumor was calculated according to the formula 0.5×length×width2. Two independent experiments were performed and each experimental group consisted of five mice. * P<0.05 vs. MKN45-pFLAG, ** P<0.01 vs. MKN45-pFLAG, # P<0.05 vs. MKN45-CacyBP.
Figure 5.
Effects of S100A6 on CayBP/SIP-mediated β –catenin degradation.
(A) Co-immunoprecipitation assay showed that truncated mutant CacyBP/SIPΔS100 bind both Skp1 and Siah1, suggesting S100A6 did not affect the formation of Siah1-CacyBP/SIP-Skp1 unbiquitin ligase complex. (B) Western blot and semi-quantitative RT-PCR were used for analysis of β-catenin protein and mRNA in transfected cells, respectively. β–catenin protein in MKN45-ΔS100 cells is significantly decreased, compared to MKN45-CacyBP cells, while with no change occurred in the mRNA. (C) The luciferase reporter gene assays were used to evaluate effect of CacyBP/SIPΔS100 on Tcf/LEF reporter activity. Transfection of wild-type CacyBP/SIP decrease the Tcf/LEF activity, compared to control plasmid. And expression of CacyBP/SIPΔS100 resulted in lower Tcf/LEF activity than wild-type CacyBP/SIP. MG132 (inhibitor of proteasome) could eliminate the effect of both wild-type CacyBP/SIP and CacyBP/SIPΔS100 on Tcf/LEF activity. *P<0.01, compared with the control; #P<0.05, CacyBP/SIPΔS100 vs. wild-type CacyBP/SIP. These experiments were repeated in triplicate.