Fig. 2
Suppression of TMEM52B promotes cancer cell invasion and survival in vitro, and tumor growth and early metastasis in vivo. (A, B) Cells were transfected with siRNA or shRNA specific to TMEM52B for 48 h. (A) Transfected cells (2 × 104 cells/well) were allowed to invade Matrigel on Transwell inserts for 48 h. The number of cells that had invaded was counted in five representative (100×) fields per Transwell insert. Knockdown of TMEM52B was analyzed by semi-quantitative RT-PCR or by immunoblot. (B) To induce anoikis, transfected cells were seeded into 96-well plates with an Ultra-Low Attachment surface and grown for up to 3 days. Cell viability was determined by the colorimetric WST assay. (C) In vivo tumor growth analysis. Stable TMEM52B-suppressed HCT-15 cells (5 × 106 cells/mouse) were injected subcutaneously into the flanks of nude mice (n = 4) as described in Materials and Methods. Tumor volume and body weight were measured for 44 days. Tumor volume was calculated using the formula, length × width2/2. Photos of tumor-bearing mice on day 44. (D, E) Ki67 (D) and TUNEL (E) staining of tumor sections from (C) was performed to measure the level of cell proliferation and apoptosis, respectively. Representative images are shown. The proliferative index (%) or apoptosis index (%) was determined by calculating the number of Ki67 or TUNEL-positive cells relative to the total number of cells (at least 1000 cells per field). Five randomly selected fields from tumor sections per mouse were analyzed. Scale bars, 200 μm. (F) Stable HCT-15 cells (5 × 106 cells/mouse) were intravenously injected into nude mice (n = 5) as described in Materials and Methods. At 24 h after injection, the lungs were removed to extract total DNA. Real-time qPCR analysis was performed for human PTGER2 using total DNA extracted from the lungs. The amount of human genomic DNA initially present in a qPCR reaction tube was estimated (top) from the standard curve generated by qPCR using human total DNA extracted from HCT-15 parental cells, which was spiked into mouse total DNA from lungs of nude mice (bottom). All in vitro determinations were performed in three independent experiments. Values represent mean ± SD. *P < 0.05. siCon, scrambled control siRNA, shCon, scrambled control shRNA; siTM, TMEM52B-specific siRNA; shTM, TMEM52B-specific shRNA