FIG 3.

Analysis of cell cycle-specific chromosome damage in HCT116 cells with or without depletion of vigilin. (A) Western blot showing depletion of vigilin after 48 and 72 h of control and vigilin-specific siRNA tranfection. (B) Cells in the plateau phase were irradiated with 3 Gy, incubated for 24 h postirradiation, and then subcultured, and metaphases were analyzed. G₁-type aberrations were examined at metaphase. All categories of asymmetric chromosome aberrations induced by IR were scored: dicentrics, centric rings, interstitial deletions/acentric rings, and terminal deletions. The frequencies of chromosomal aberrations were identical for cells with or without depletion of vigilin, indicating normal repair of G₁-phase-specific chromosome damage. (C) Dicentrics, which formed mostly in G₁, were analyzed after 3 Gy of IR exposure, and no differences were found in cells with and without depletion of vigilin. (D) Cells in the exponential phase were irradiated with 2 Gy. Metaphases were harvested at 4 h after irradiation, and S-phase types of chromosomal aberrations were scored. Cells with vigilin knockdown showed significant differences in chromosomal aberration frequencies compared to control cells. (E) Radials were analyzed after IR exposure. (F) Cells in the exponential phase were irradiated with 1 Gy. Metaphases were harvested following irradiation, and G₂-type chromosomal aberrations were monitored. Cells with vigilin knockdown showed modest increase in frequencies of chromosomal aberrations compared to control cells. Each experiment was repeated three times, and for each experiment, 300 metaphases were scored.