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. Author manuscript; available in PMC: 2011 Apr 1.
Published in final edited form as: Cancer Res. 2010 Mar 16;70(7):2942–2950. doi: 10.1158/0008-5472.CAN-09-3826

Figure 3. Overexpression of LZTFL1 in Hela cells inhibits anchorage-independent cell growth in vitro.

Figure 3

(A) Dox-regulated production of LZTFL1 in stable Hela-tet-on-LZTFL1 clones. Clones 10, 29, and 32 were generated by transfecting a pTRE2 plasmid containing a Dox-responsive expression cassette that encodes Flag-LZTFL1 and a EGFP reporter into Hela-tet-on cells. Cells were exposed to various amounts of Dox as indicated for 24 hrs, and whole cell extracts were analyzed by immunoblotting using anti-Flag antibody. (B) The colony-forming ability of the parental, EGFP-expressing, and LZTFL1-expressing Hela-tet-on cells in soft agar in the presence and absence of Dox was examined as a measure of anchorage-independent growth. 2000 cells were seeded and colonies were photographed four weeks after plating (upper panel, representative ones) and counted (lower panel, n=3 ± SEM). (C) Soft agar assays for HT-29 cells expressing EGFP or LZTFL1. HT-29 cells were transfected with control vector pEGFP and pEGFP-ires-LZTFL1, respectively. Stable clones (vector, 1, 2, and 3) were selected and expanded in the presence of G418. 400 cells from clones indicated were seeded for soft agar assays. Colony numbers were counted (n=3 ± SEM). (D) Transwell migration assays with Hela-tet-on, EGFP, LZTFL1-32 and LZTFL1-10 cells previously cultured in the absence and presence of Dox (n=3 ± SEM). Induction of LZTFL1 inhibited the migration of Hela cells in both LZTFL1-32 and LZTFL1-10 clones.