Figure 1. GARP expression in human breast cancer and its oncogenic roles in murine mammary gland epithelial cells.

(A) IHC validation of the human GARP antibody using murine 4T1 cells expressing human GARP (hGARP). Upper panels: staining of 4T1 cells expressing hGARP with anti-hGARP or control antibody. Lower panels: hGARP antibody was pre-adsorbed with 10 μg of recombinant hGARP (rhGARP) or 10 μg of bovine serum albumin (BSA) for 30 minutes at room temperature prior to the application on the slides. Scale bar: 40 μm. (B) Representative images of GARP IHC (brown color) of breast cancer with their matched normal breast tissues. Each patient specimen in these TMAs was represented in two cores on the slide and each core measured 1 mm in diameter. Scale bar: 50 μm. (C) Expression intensity of GARP-positive cells in breast cancer specimens and normal breast tissues. (D) shRNA knockdown of GARP mRNA in NMuMG* cells. Cells treated with scrambled shRNA (SCR) were used as control. Real-time RT-PCR was performed to quantify mRNA of Lrrc32 using β-actin as a control. MNE: mean normalized expression. (E) Flow cytometric analysis of cell surface GARP expression on GARP KD and SCR NMuMG* cells. (F) Immunoblot of GARP and TGF-β level in GARP KD and SCR NMuMG* whole cell lysates. (G) In vitro cell proliferation assay for GARP KD and SCR NMuMG* cells by MTT assay. (H) NMuMG* SCR and NMuMG*-GARP KD cells were injected into NOD-Rag-1^−/− mice, followed by weekly monitoring of the tumor growth kinetics. (I) 120 days after tumor injection, lungs gross metastatic nodules were counted visually on the surface of the organ. **P<0.01. Statistical analysis was determined by 2-way ANOVA or two-tailed T-test, where appropriate. Data are representative of at least two independent experiments.