PDLIM2 expression is repressed in colorectal cancer cell lines. A, Protein expression of PDLIM2 in the indicated cell lines was analyzed by immunoblotting (IB) using a PDLIM2 specific antibody. B, The relative levels of PDLIM2 mRNAs in the indicated colon cancer cell lines were analyzed by real-time PCR using β-actin mRNA as a control. The data are represented as the mean percent of expression observed in control MCF10A cells ± standard deviation (n = 3).

PDLIM2 repression in colon cancer cells involves DNA methylation. A, RNA levels of DNMT1, DNMT3a and DNMT3b in the indicated colon cancer cells were analyzed by real-time PCR using β-actin mRNA level as a control and represented as fold induction in mRNA abundance relative to those in MCF10A cells (set as 1). The data presented are the mean ± standard deviation (n = 3). B, The indicated cell lines were treated with the DNMT inhibitor 5-aza-dC (5μM) for 48 h, followed by real-time PCR to determine relative mRNA levels of PDLIM2. Changes in PDLIM2 mRNA abundance following 5-aza-dC treatment are represented as fold induction relative to those observed in an RNA sample from mock-treated cells. C, The indicated colon cancer cell lines were treated with 5 μM 5-aza-dC or vehicle for the indicated time points, followed by cell growth assay. D, The indicated cell lines were treated with 5 μM 5-aza-dC or vehicle for 5 days, followed by the bisulfite genomic DNA sequencing as described in Material and Methods. Each circle represents a CpG site; open circles represent unmethylated CpG dimucleotides whereas filled circles represent methylated CpG sites. The ratios of the filled area in circles represent percentiles of the methylation in the CpG sites. The position of each CpG nucleotide relative to the PDLIM2 transcription initiation site (+1) is indicated at the top.

PDLIM2 inhibits NF-κB constitutive activation in colon cancer cells. A–D, The indicated colon cancer cell lines were transfected with κb-TATA driven luciferase reporter in the presence of increasing amounts of Myc-PDLIM2, followed by luciferase assay. The luciferase activities were presented as the percentile of that in cells without Myc-PDLIM2 transfection (denoted as 100). The protein expression levels of Myc-PDLIM2, p65 and Hsp90 were detected by direct immunoblotting assays and the non-specific bands were indicated by the asterisks.

PDLIM2 re-expression suppresses tumorigenicity of colorectal cancer cell lines. A & B, DLD1 and HCT116 colon cancer cells stably expressing Myc-PDLIM2 or an empty vector were plated in soft agar and colony numbers were counted at day 21 after plating. The data presented are the mean ± standard deviation (*p<0.01). C & D, The DLD1 and HCT116 stable cell lines were subcutaneously inoculated into the hind back of the SCID mice. The mice were sacrificed at day 14 after inoculation and tumor weights were measured. The data presented are the mean ± standard deviation (**p<0.05).