Fig 2: COL5A2 gene and protein expressions are prognostic markers of CRC. (A) Kaplan-Meier analysis of the overall survival (OS) and disease free survival (DFS) of CRC patients based on COL5A2 gene expression using Smith Colorectal dataset (GSE17536) from Oncomine database.27 The low or high expression of COL5A2 was defined as lower-than the median (= median) or higher-than the median (> median). The case numbers of each group are indicated in the blankets. The p value was calculated using the Log-rank (Mantel-Cox) test. (B) COL5A2 protein expression was analysed in CRC using two commercial TMAs containing 180 CRC and paired normal tissues. The “full tissue spot”, the “stromal” region and the “epithelial” region were defined manually and the staining intensities were calculated automatically using the Positive Pixel Count algorithm v9.1 with Aperio ImageScope. The Positivity value was used to represent the expression level of COL5A2 protein. The optimal cutoff to dichotomize the cases was determined dynamically using X-title v3.6.1.17

Fig 3: Expression of COL5A2 protein in colon cancer tissues. Immunohistochemistry (IHC) analysis was performed using two commercial tissue microarrays (TMAs) containing a total of 180 cases. (A) The image shows the negative or low expression of COL5A2 in normal colonic mucosa tissue. The bar represents 500 µm. The boxed area is enlarged to the right side. (B) Low and (C) high expression of COL5A2 in CRC tissue. (D) A statistic of COL5A2 expression between the normal and cancer tissues based on the scanned staining intensity values.

Fig 4: Ectopic expression of COL5A2 promotes cancer cell proliferation and upregulates WNT/ß-catenin and PI3K/mTOR pathway. (A) COL5A2 was ectopically expressed in HEK293T and colon cancer cell lines SW620 and HCT116 and the cell variability was measured using MTT method in different days. (B and C) Ectopic expression of COL5A2 and selected signaling molecules were measured by Western blot. (D) Western blot analyses of DDR1 and pY792 DDR1 in COL5A2 overexpressing CRC cells. The Western blot densitometry was calculated using Multi Gauge V3.0 and the fold change was normalized using the blank vector transfection. COL5A2 protein was probed using two antibodies. Active ß-catenin was the endogenous ß-catenin protein when residues Ser33, Ser37, and Thr41 are not phosphorylated. The arrow heads indicate the positions of GSK3ß.