Journal: Cancer Biology & Therapy
Article Title: FOXQ1 mediates the crosstalk between TGF-β and Wnt signaling pathways in the progression of colorectal cancer
Figure Lengend Snippet: Knockdown of FOXQ1 partially inhibited TGF-β1-induced aggressive tumor behaviors in SW480 cells and the changes in expression of downstream genes. ( A ) NC- and FOXQ1-shRNA cells were incubated in growth media supplemented with TGF-β1 (3 ng/ml) for 3 days. FOXQ1 expression was measured by qRT-PCR and Western blot. ( B ) The culture supernatant from NC-shRNA cells cultured for 3 days or NC- and FOXQ1-shRNA cells cultured for 5 days containing TGF-β1 (3 ng/ml) was collected and used in an in vitro angiogenesis assay. The vessel formation was measured in NC-shRNA-transduced EA.hy926 cells. The number of vessels was counted by fluorescent microscopy (120X). ( C ) FOXQ1- and NC-shRNA cells were treated with TGF-β1 (3 ng/ml) for 5 days, and cell morphology was examined by optical microscopy (120X) (upper panel). For the invasion assay, FOXQ1- and NC-shRNA cells were pre-treated with TGF-β1 (3 ng/ml) for 3 days before being seeded in the upper chamber of Transwell. After 36 hours, the cells migrating through the insert membrane were fixed and stained by crystal violet. The number of migrated cells was counted by optical microscopy (120X) (lower panels). ( D ) For the drug sensitivity assay, FOXQ1- and NC-shRNA cells were incubated in TGF-β1-containing media (3 ng/ml) for 4 days, and 5-FU or L-OHP (100 μg/ml) was added to the cell culture for an additional 24 hours. Flow cytometry was performed to measure cell apoptosis. ( E ) Expression of Wnt downstream target genes and EMT-associated markers in TGF-β1 treated NC- and FOXQ1-shRNA cells were measured by qRT-PCR and Western blot. The results are expressed as mean ± SD *P
Article Snippet: All reagents and primers used in quantitative real-time polymerase chain reaction (qRT-PCR) were purchased from Takara Bio (Dalian, China).
Techniques: Expressing, shRNA, Incubation, Quantitative RT-PCR, Western Blot, Cell Culture, In Vitro, Angiogenesis Assay, Microscopy, Invasion Assay, Staining, Sensitive Assay, Flow Cytometry, Cytometry