CD74 is a survival receptor on colon epithelial cells

Figure 5 CD74 is expressed on CT26 cells and regulates their survival. A: CT26 cells were stained with anti CD74 antibodies, and analyzed by FACS. Histogram shows CD74 expression on these cells; B: Serum-starved CT26 cells were incubated in the presence or absence of MIF (400 ng/mL) for 6 h. Histograms show FLICA staining for apoptotic cells in both groups; C: Graph representing the average of five different experiments, demonstrating decreased apoptosis in MIF treated compared to untreated CT26 cell line; D: Serum-starved CT26 cells were either kept untreated or treated with MIF (400 ng/mL) for 3 h and levels of Bcl-2 mRNA were analyzed by RT-PCR. The results presented are representative of five different experiments; E: Quantitative real time PCR was performed using primers for Bcl-2. β-actin levels were used to normalize samples for calculation of the relative expression of Bcl2 level. Results are expressed as a fold of change in Bcl-2 expression at stimulated cells compared to non stimulated cells, which was defined as 1. Results shown are a summary of four separate experiments; F: Following 6 h of starvation the CT26 cells were lysed and Bcl-2 protein expression was analyzed by Western blotting analysis; G: Quantitative real time PCR was performed using primers for cyclin E and β-actin. β-actin levels were used to normalize samples for calculation of the relative expression levels of cyclin E. Results are expressed as a fold of change in cyclin E expression at stimulated cells compared to non stimulated cells, which was defined as 1. Results shown are a summary of four separate experiments; H: CT26 cells were incubated in the presence or absence of MIF (400 ng/mL) for 1 min. Cells were fast-frozen and lysed as described in Methods. Lysates were separated on 10% (wt/vol) SDS/PAGE and blotted with anti-p-Akt antibody (results presented are representative of three separate experiments) followed by HRP-conjugated anti-mouse antibodies. The membranes were then stripped and blotted with anti-tubulin.