Figure 2. Cell cycle and colony formation analysis in selected Nutlin-Resistant clones.
A. Selection of Nutlin-Resistant SJSA-1 cell clones. P4 populations from Experiments 1-4 were plated in the absence or presence of Nutlin for 14 days. Individual colonies were then isolated and expanded. Group 1 clones (isolated in the absence of Nutlin) undergo growth arrest in the presence of Nutlin but are resistant to Nutlin-induced apoptosis. Group 2 clones (isolated in the presence of Nutlin) are resistant to both apoptosis and growth arrest induced by Nutlin. The p53 status of the Group 1 and Group 2 clones from each experiment that were used in subsequent experiments was determined by cDNA sequencing and is indicated. The p53 sequence of all clones isolated in each experiment is listed in Supplemental Table 1. B, D. SJSA-1 (SJ) and Nutlin-resistant Group 1 (G1) and Group 2 (G2) clones were untreated or treated with 10 μM Nutlin for 72 hrs and subjected to FACS analysis. The percentage of cells with sub-G1 DNA content (B) and cell cycle distribution (D) were determined from DNA profile histograms using FlowJo. U: untreated; N: Nutlin treated. C. SJSA-1 (SJ) and Nutlin-resistant Group 1 and Group 2 clones were plated at low density (1×102 - 1×105 cells / 10cm dish) and either untreated or treated with 10 μM Nutlin for 14-21 days. Colony forming ability was determined as described in Figure 1.