Figure 3
Cbx7 affects the different tumorigenic properties of glioma cells.
(A) U373 cell line was transfected with pCMV-Vector and pCMV-Cbx7 and selected for G418 resistance for two weeks. The stable U373/VC and U373/Cbx7 were harvested for RNA and protein extraction. The column plot indicates Cbx7 transcript levels, as assayed by qRT-PCR. (B) U373/VC and U373/Cbx7 stables were counted and plated at a density of 2.5 × 103 cells per well and allowed for colony formation. Colonies were stained with crystal violet. Mean colony counts are displayed as percentage of vector control. (C) Control Vector and Cbx7 overexpressing stables of U373 cell line were counted and plated (4.0 × 104 cells) for proliferation in duplicates. Cells were counted every third day using viability cell counter. The column plot indicates the percent cell number at the respective time intervals. (D,E) U373 cell line was transfected with pCMV-Vector and pCMV-Cbx7, after 48 hours cells were harvested for cell cycle analysis by propidium iodide staining, the numerals (bold) represent the percent sub-G1 population (apoptotic cells). The column plot depicts the quantitation of sub-G1 cell population from three independent experiments. Percent sub-G1 population with standard deviation is plotted. (F) The western blot indicates more of cleaved caspase-3 in Cbx7 overexpression condition, indicating more apoptosis. (G) U373/VC and U373/Cbx7 stable cells were subjected to in vitro scratch assay, the area of scratch was photographed at the time of scratch (0th hour) and after regular intervals to monitor wound closure. The column plot represents the gap of the wound (average of 8 different locations from two different wells). (H,I) U373/VC and U373/Cbx7 stable cells were plated for Boyden chamber migration assay and matrigel invasion assay. After 22 hours, cells were fixed, stained with crystal violet and photographed. The column plot represents the number of cells migrating or invading respectively, cell number with standard deviation is plotted. Significance testing was performed between U373/VC and U373/Cbx7 in different experiments using student’s t-test and the symbols are indicated. *p ≤ 0.05; **p ≤ 0.01 and ***p ≤ 0.001.
