Figure 3
Sp1 negatively regulates invasive and migratory abilities of lung cancer cells. (a) CL1-5 cells were infected with adenovirus-GFP and increasing doses (1–20 m.o.i.) of adenovirus-GFP-Sp1. After incubation for 48 h, cells were harvested for whole-cell lysates and cellular proteins were immunoblotted with anti-Sp1, CCSP and actin antibodies. pSp1 represents phosphorylated Sp1. (Lower panel) The quantitated result for the ratio of exogenous GFP-Sp1 with endogenous Sp1, both of which were normalized by β-actin. (b) The morphologies of adenovirus-GFP- and GFP-Sp1-infected CL1-5 cells were observed using light microscope with × 100 magnification. (c) Effect of Sp1 on invasive ability. The in vitro invasive ability of CL1-5 cells infected with adenovirus-GFP or GFP-Sp1 was determined using Matrigel-combined Transwell chambers as described in Materials and methods. Data are representative of three independent experiments and are presented as mean±s.e.m. (*P<0.05). (d) Effect of Sp1 on the migratory ability of cells measured with a wound-healing assay. After infection with adenovirus-GFP or GFP-Sp1 for 48 h, confluent monolayers of CL1-5 were wounded with a pipette tip and incubated for an additional 24 h. The migratory area of cells was calculated for quantification. Data are representative of three independent experiments and are presented as mean±s.e.m. (*P<0.05). (e) Effect of Sp1 on migratory ability examined by a Transwell migration assay. After infection for 48 h, the migratory ability of adenovirus-GFP or GFP-Sp1-infected cells was determined using Transwell chambers as described in Materials and methods. Data are representative of three independent experiments and are presented as mean±s.e.m. (*P<0.05). (f) Effect of Sp1 on migratory ability monitored by video time-lapse microscopy. After infection with adenovirus-GFP or GFP-Sp1 for 48 h, the migratory area of CL1-5 cells was continually monitored for 24 h under a time-lapse microscopy at × 100 magnification.
