Figure 7

Impact of rat anti-AGR2 Ab on cell growth and cyclin D1 in T47 D cells. (a) Rat anti-AGR2 Abs were tested for AGR2 specificity by using an ELISA directed against human AGR2 and human AGR3. Species crossreactivity also was assessed by using an ELISA directed against mouse AGR2. (b) After confirming Ab specificity, T47 D cells were treated with an anti-AGR2 Ab (10 μg/mL) for 48 hours or AGR2 siRNA for 72 hours, and cyclin D1 modulation was examined with immunofluorescence. Cells were stained with cyclin D1, and mounting media containing DAPI were used. Images were taken by using a fluorescence microscope and pseudo-colored in Adobe Photoshop. The isotype control Ab used for cyclin D1 staining was an anti-AGR2 Ab of the same isotype but was not shown to modulate cyclin D1 or to have an impact on growth. Cyclin D1 intensity was quantitated by using ImagePro and binned based on intensity, and the percentage of cells in each bin based on cyclin D1 intensity is represented (Bin 1, weakest staining; Bin 4, brightest staining). (c) T47 D, ZR-75-1, and MDA-MB-231 cells were treated for 5 days with 20 μg/mL anti-AGR2 Ab. The relative number of cells was quantitated by using the MTT assay. Results are expressed relative to untreated sample for each cell line.