Figure 6. Genomic instability caused by ATR suppression is significantly enhanced by H-ras^G12V expression.

(A) Western blot analysis of immortalized MEF cultures 48 hours following initial 4-OHT treatment. Separate blots from similarly prepared protein lysates were detected for ATR and MCM3 (top panels) or γH2AX, phospho-ERK, and actin (bottom panels). S-phase content was quantified through EdU incorporation/detection in cultures harvested 48 hours after initial 4-OHT treatment. Cumulative doublings were obtained through cell counting at 48 hours after initial 4-OHT treatment. (B) Chromosomal breakage analysis of MEF cultures from A. Cultures were collected for metaphase analysis 48 hours after initial 4-OHT treatment. Gaps/breaks per metaphase were scored in readily assessable metaphase spreads, while metaphase spreads with more than 15 breaks were scored separately and weighted into this analysis. The percentage of metaphase spreads with more than 15 breaks is listed in the lower panel. PCC was also scored, and the percentage of metaphase spreads marked by PCC is listed in the lower panel. Data represent mean ± SEM.