Figure 1.

TREX1 silencing inhibits proliferation, clonogenic and anchorage independent growth of cervical cancer derived cell lines. (A) The levels of TREX1 were determined by western blot using 30 μg of total protein extracts from monolayer cultures of primary human keratinocytes (PHK), PHK transduced with HPV11 (PHK pBabe, PHK pBabe 11E6E7) or HPV16 (PHK pLXSN, PHK pLXSN 16E6E7) genes and cervical cancer derived cell lines C33A, SiHa (HPV16) and HeLa (HPV18). (B) The expression of TREX1 in C33A, SiHa and HeLa cells was silenced using lentiviral particles expressing specific shRNAs. Silencing efficiency was determined by western blot as described in A. (C) For proliferation assays (growth curves) 5000 cells of each cell line were seeded in 6-well plates and counted daily for seven days. (D) For clonogenic assays 1000 cells of each cell line were seeded in 100 mm Petri dishes and cultured for 15 days. Colonies were stained with crystal violet and counted. (E) For anchorage independent growth 500 cells of each cell type were seeded in 24 well plates in 0,6% agarose prepared in M10. After 30 days colonies were stained with MTT and counted. The results shown are representative of at least three independent experiments performed in triplicate. *p-value ≤ 0.05. Western blot signals were quantified using ImageJ software using housekeeping genes actin or tubulin as normalizers and presented as expression relative to normal keratinocytes.