Figure 4. ATR reduction potently suppresses the growth of p53-null fibrosarcomas driven by H-ras^G12V and increases genomic instability.

(A) Quantification of correctly spliced mouse (ATR^fl) and human (ATR^seckel) ATR transcript in p53^–/–ATR^fl/seckelCreERT2⁺ cell lines expressing H-ras^G12V. Relative transcript quantity was measured through qRT-PCR analysis of RNA isolated from the indicated cultures (n = 4 independent cell lines). Data represent mean ± SEM. (B) Representative images of tamoxifen-treated ATR^fl/+CreERT2⁺ (top panel) or ATR^fl/seckelCreERT2⁺ (bottom panel) tumors 10 days following the initiation of treatment. TAM, tamoxifen. (C) Measurement of fibrosarcoma growth following ATR^fl deletion. Tumors were allowed to grow to 100–200 mm³ before initiation of tamoxifen treatment to recombine the ATR^fl allele. Tamoxifen treatment days are indicated by arrows. *P < 0.01 (n = 4–13 tumors per genotype, per measurement). Percentages listed above at specific time points indicate the abundance of the ATR^fl-recombined allele (ATR^Δ) in tumors isolated from that time point (n = 1–3 tumors per genotype/measurement). Data represent mean ± SEM. (D) H&E-stained sections (left panels) or γH2AX/DAPI–stained sections (right panels; γH2AX, green; DAPI, blue) of H-ras^G12V–expressing tumors (A–C) isolated 10 days after initial ATR^fl deletion. Enlarged cells with aberrant nuclei are indicated with arrows. Original magnification, ×200. (E) Quantification of γH2AX-positive cells in H-ras^G12V–expressing tumors (A–C) isolated 9–10 days after initial ATR^fl deletion. The abundance of γH2AX-positive cells was determined from 10 high power field images (HPF, ×200) from each tumor type (n = 4–6 tumors per genotype). Only nucleated (DAPI-positive) cells were scored in this analysis. Data represent mean ± SEM.