Figure 1. CCDC6 knock down alters proliferation rate and increases cell death in vitro.

Cells were transduced using lentivirus, expressing two different small hairpins for CCDC6, labeled as sh1 and sh2 and cultured for 48 hours. The same viral vector was applied as control (mock: mock transduced). (A) Western blot analysis using anti-CCDC6 mouse monoclonal antibody demonstrated the efficient knock down of CCDC6 protein expression. Growth curves were performed in triplicates using trypan blue dye exclusion for counting the alive (B) and the dead cells (C). Decreased proliferation rate and increased cell death was observed in the absence of CCDC6. (D) The subG₀/G₁ population, as measured by flow cytometry, is indicative of apoptosis and is significantly increased following CCDC6 knock down. The percentage of survival was calculated for each time point by excluding both early apoptotic and dead cells. (E) Apoptotic cell death was analyzed by Po-PRO and 7-ADD staining. The Po-PRO single-positive cells are early apoptotic while the double positive stained cells for Po-PRO and 7-AAD are late apoptotic and dead cells. All assays were performed in three independent experiments.