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. Author manuscript; available in PMC: 2011 Apr 2.
Published in final edited form as: Cell. 2010 Apr 2;141(1):69–80. doi: 10.1016/j.cell.2010.02.027

Figure 2. Heterogeneity and reversibility in the drug-tolerant cell populations.

Figure 2

(A) PC9 cells and PC9-derived DTPs were processed for immunofluorescence using anti-CD133 and counterstained with Hoechst to visualize nuclei. Magnification, 20X.

(B) Cell lysates from PC9 cells and PC9-derived DTPs were analyzed by immunoblotting with anti-CD133, and anti-GAPDH as loading control.

(C) PC9 cells and PC9-derived DTPs were labeled with CD24 antibody conjugated to PE and a CD44 antibody conjugated to APC and analyzed by FACS. Note the enrichment of cells with surface expression of CD24 in DTPs (quantitation is shown in Fig. S4).

(D) Uncloned PC9 cells or cells from two different single cell-derived PC9 clones (A and B) were treated with 2μM erlotinib for 33 days and then Giemsa stained.

(E) Survival curves of PC9 cells and PC9-derived DTPs following recovery and re-expansion in drug-free medium and subsequent exposure to the indicated erlotinib concentrations for 72 hours, demonstrating the reversibility of drug tolerance. Each data point represents the average value determined from four samples. Data are expressed as percent surviving cells relative to untreated controls. Error bars represent standard deviations from the mean. The dashed line corresponds to 50% cell killing.

(F) Survival curves of PC9 cells and a PC9-derived DTEP line, GR7, after gefitinib withdrawal for the indicated number of passages (P) and subsequent exposure to the indicated gefitinib concentrations for 72 hours. Each data point represents the average value determined from four samples. Data are expressed as percent surviving cells relative to untreated controls. Error bars represent standard deviations from the mean. The dashed line corresponds to 50% cell killing.

(G) PC9 cells or PC9-derived DTEPs after gefitinib withdrawal for the indicated number of passages were either untreated or treated with 2 μM gefitinib (GEF) for 48 h, after which cells were subjected to FACS analysis. Presented in the graph is the percentage of dying cells in the population (sub-G1 DNA content). Error bars represent standard deviations from the mean from duplicate plates.