(A) Left, HCC827 (upper) or PC9 (lower) cells were treated with 0.1 µM erlotinib for 24 hr, followed by immunofluorescence staining using an antibody to SOX2 and DAPI. For erlotinib-treated cells (middle and right pairs of images), the heterogeneity in induced SOX2 levels per cell in each population is indicated by dashed outlines indicating DAPI+ nuclei lacking SOX2, white arrows for nuclei with low (but detectable) SOX2 and green arrows for nuclei with high SOX2. Right, distribution of SOX2 fluorescence in each sample. Mean fluorescence counts for each cell were quantitated and normalized to exposure time as described in ‘Materials and methods’. p < 0.0001 for the comparison of erlotinib-treated cells to DMSO (Student's t-test, unequal variances, N = 1219–3485, means are 0.005/0.04 for untreated/treated HCC827 and 0.001/0.008 for untreated/treated PC9, % SOX2+ is shown). Source data are included as Figure 2—source data 1, 2. (B) Induction of SOX2 in erlotinib-retreated cells. HCC827 cells were treated with 1.0 µM erlotinib for 24 hr (75% cell killing), followed by removal of drug, replating of cells after 7 days of recovery and retreatment with the same concentration of erlotinib. This protocol was repeated, and then cells were treated a third time with erlotinib for 24 hr and analyzed by immunofluorescence microscopy using antibodies to SOX2 and DAPI. The increase in SOX2-positive cells was highly significant for both erlotinib pretreated and untreated cells, but no enrichment was observed as a consequence of pretreatment p < 0.0001 for the comparison of erlotinib-treated cells to DMSO (Student's t-test, unequal variances, N = 1106–2143, means are 0.005/0.17 for untreated/treated-parental, 0.007/0.2 for untreated/treated-pretreated, % SOX2+ is shown). Source data are included as Figure 2—source data 3. (C) Immunofluorescence analysis of single colonies formed from single clones of HCC827 cells stained for DAPI (blue), SOX2 (green), and Ki67 (red).