Figure 5
L1-attenuated glioma cells were less motile. (A) 9L/LacZ cells were infected with mouse antisense-L1 retroviral vector or control pLEGFP-C1 vector. The positively infected cells were then sorted by FACS. Post-sort analysis of live cells is shown with fluorescence intensity level displayed along the X-axis. The black peak represents uninfected cells, the blue line represents pLEGFP-C1 vector infected negative control cells (96% infected), and the red line represent the cells infected with antisense-L1 vector (87% infected). Antisense-L1 infected cells were less bright green than the extremely bright control GFP-only cells, presumably because of fusion of GFP to the antisense sequences. (B) L1 expression was analyzed by western blot in vector infected 9L cells purified above. Whole cell lysates were made and the same amount of total protein was loaded into each lane (30 μg). Western blots were stained with NCAM-L1 polyclonal anti-cytoplasmic L1 and the density of the 32 kDa cytoplasmic L1 fragment was analyzed using Un-Scan-It software. The 32 kDa cytoplasmic fragment was reduced by approximately 90% in the antisense infected 9L/ASL1 cells compared to the 9L/GFP negative control cells. Blots were probed for GAPDH, which was used to normalize protein levels for quantitation of L1. (C) Cells were assessed by the Super Scratch assay for effects on cell motility. Antisense-L1 infected 9L/lacZ cells showed decreased motility (red graph) compared to pLEGFP infected control cells (blue graph). The time-lapse graph shows the average velocities (thin lines) and 3rd order polynomial best-fit curves (thick lines) of control and antisense-L1 infected populations of cells analyzed above at 15 min. intervals. The average velocity of the antisense-infected cells was consistently lower throughout the time course of the experiment. (D) The overall average velocities were calculated using all individual cell velocities collected during the course of the experiment and graphed as single values for the two populations of cells. Control GFP-only infected cell velocity was 0.381 ± 0.012 s.e.m. and AS-L1 infected cell velocity was 0.253 ± 0.008 s.e.m. The differences in the velocities are highly significant (p < 0.001). The experiment was repeated twice with similar results.