Article Figures & Data
Figures
- FIGURE 1.
Effects of the Src inhibitors on glioma invasion. A. Dose-dependent decrease in the invasion of U251 spheroids in a three-dimensional collagen matrix with PP2 but not with PP3 (n = 8 spheroids per condition). **, P < 0.01, compared with control (two-factor mixed-design ANOVA). B. Similar effect observed with SU6566 (SU). C. Single-cell invasion distances of U251 glioma cells between 24 and 48 hours after implantation measured using time-lapse videomicroscopy. Measurements were separated in 100 μm intervals. Bars, % cells whose invasion distance fell within that interval in each experiment. D. Effect of addition in the matrix of 10 μmol/L PP2 on the distribution seen in C. The great majority of the cells (70%) do not invade >50 μm in that interval (n = 3 movies, an average of 40 cells measured for each). **, P < 0.01, significantly higher than control; ††, P < 0.01, significantly lower than control.
- FIGURE 2.
U251 glioma cells express Src, Fyn, and Yes but not N-Src. A. Western blot (WB) of total cell lysates obtained from 293 cells, normal human brain, and U251 cells probed for Src (top). Immunoprecipitation (IP) of the same lysates using a N-Src-specific antibody probed with the Src antibody, which recognizes both N-Src and c-Src, showing that N-Src is expressed in normal human brain but not in the human glioblastoma cell line U251 (bottom). B. Western blot of total cell lysate from U251 cells probed for Yes and Fyn antibodies. C. Efficient inhibition of Src activity by the inhibitor PP2. Total cell lysates of U251 cells expressing activated Src were probed with an anti-phosphotyrosine antibody to confirm the disappearance of the signal when increasing concentrations of PP2 are added.
- FIGURE 3.
Effects of PP2 on U251 glioma cells proliferation. A. Decrease in the proliferation rates of glioma cells by PP2 and SU6656. At day 0, 50,000 cells were plated in six-well plates and the inhibitors were added at day 1 (arrow). Cells were counted every 2 days by trypsinizing the cells and feeding them in a Coulter particle counter. **, P < 0.01, compared with control (two-factor ANOVA). B. Decrease in the proliferation index of the cultures with PP2 but not with PP3 as measured by the ratio of Ki67-positive nuclei 4 days after addition of the inhibitors. **, P < 0.01, compared with control (ANOVA).
- FIGURE 4.
Expression of DN-Src and CSK in the cells invading the collagen matrix. A. Infection of the cells with a GFP-expressing adenovirus (bottom) did not significantly alter their invasion velocity compared with control, noninfected cells in the same spheroids (top). B. Expression of a DN mutant of c-Src significantly decreased U251 invasion distances. C. Expression of CSK also, to a lesser extent, shifted the distribution toward smaller invasion distances (n = 3 movies, an average of 20 cells measured for each). **, P < 0.01, significantly higher than control; †, P < 0.05; ††, P < 0.01, significantly lower than control.
- FIGURE 5.
Effect of PP2 on peripheral membrane ruffles in glioma cells. A. Fluorescent time-lapse videomicroscopy images of a single U251 stably expressing a YFP-actin construct before and after addition of 10 μmol/L PP2 show a selective disappearance of the peripheral membrane ruffle (arrow). Bar, 20 μm. B and C. Quantification of this effect using low-magnification time-lapse videomicroscopy of untransfected U251 cells. Under phase, the peripheral lamellipodia appear as phase-dark ruffles (arrows) and disappear after addition of PP2 (B). Bar, 50 μm. Quantification shows that this effect is specific to PP2 and becomes significant 2 minutes after addition of PP2 at time 0 (n = 8 movies, an average of 40 cells counted for each). **, P < 0.01 compared with PP3 (two-factor mixed-design ANOVA). D and E. Effects of the recombinant adenoviruses on the number of cells with membrane ruffles in the population (n = 3, an average of 95 cells counted for each). *, P < 0.05; **, P < 0.01, compared with control (ANOVA). Images show typical fields 24 hours after addition of the virus. Bar, 50 μm.
- FIGURE 6.
Effect of PP2 on the actin dynamics in U251 in the three-dimensional collagen matrix. A. YFP-actin image of a cell invading away from the spheroid (bottom). Bar, 20 μm. B. High-magnification zoom of the invading tip of the cell showing a “burst” of actin. Bar, 2 μm. C. Quantification of the fluorescence intensity in the area in A shows that this bursting occurs at regular interval in this cell and is abrogated by the addition of 5 μmol/L PP2 on the collagen gel (dashed line).
Tables
- Table 1.
Summary of the Mean and Maximum Distances (μm) Obtained in Figs. 1C and D and 4
Mean ± SD Invasion (μm) Maximum Invasion (μm) P Control (n = 281) 198 ± 192 884 PP2 (10 μmol/L, n = 68) 43 ± 60 333 <0.01 Ad-GFP (n = 82) 175 ± 174 771 NS Ad-DN-Src (n = 30) 44 ± 60 250 <0.01 Ad-CSK (n = 44) 90 ± 85 323 <0.01 NOTE: Maximum invasion represents the furthest distance recorded for a single cell in the invading population for a 24-hour period. Ps refer to the comparison of the mean invasion values by ANOVA. See text for details.
Volume 2, Issue 11
