ActD treatment induces p53-dependent cell death in Eμ-Myc lymphomas in vivo. (A) C57BL/6J and C57BL/6J-Ly5.1 mice were intravenously injected with 200 000 Trp53^+/+;Eμ-Myc or Trp53^–/–;Eμ-Myc lymphoma cells, respectively, and were treated with a single dose of 0.1 mg/kg ActD or vehicle. Spleen, inguinal, and axillary lymph nodes were collected 6 hours after drug administration from 3-5 mice per group as described in supplemental Methods. (B-C) qRT-PCR analysis of Its1-containing 47S pre-rRNA (B) and of Puma, Noxa, and p21 (C) in axillary nodes from ActD-treated mice, normalized to β2m mRNA levels and compared with vehicle-treated axillary nodes. *P < .05; ***P < .001; ****P < .0001 (unpaired Student t test). (D-E) Percentage of viable lymphoma cells in the spleen of Trp53^+/+;Eμ-Myc (D) or Trp53^–/–;Eμ-Myc (E) lymphoma-bearing mice determined by flow cytometry as the percentage of PI-, B220⁺ (pan B-cell marker), and either green fluorescent protein (GFP⁺) for Trp53^+/+;Eμ-Myc lymphoma cells or CD45.2⁺ cells for Trp53^–/–;Eμ-Myc lymphoma cells, as detailed in supplemental Methods. Data are presented as individual values with error bars displaying mean ± SEM. *P < .05 (unpaired Student t test). (F) Western blot analysis of the spleen homogenates of each Trp53^+/+;Eμ-Myc lymphoma-bearing mouse analyzed in panel D. Expression of the 2 MCL-1 forms (upper and lower bands) as determined in Figure 2A is shown as relative to MCL-1 expression in the vehicle-treated group. Data are presented as individual values with error bars displaying mean ± SEM. *P < .05 (2-way ANOVA test). a.u., arbitrary units; d0, day 0.