(A) Cell lines were treated with an inhibitor of the driving oncogenic lesion for 24 hr (erlotinib for EGFR-mutant, lapatinib for HER2-amplified and crizotinib for ALK-translocated cells), followed …
(A) Heat map showing the list of fourfold significantly (FDR <0.05) upregulated (green) or downregulated (red) transcripts in HCC827 cells with erlotinib treatment. (B) Time course of changes in …
(A) HCC827 and PC9 cells. The erlotinib curve is the same as in Figure 1B. (B) Left panel, H1650 cells (EGFR DEL15 activating but IC50 >1 µM) and WM164 (BRAF V600E) show minimal induction following …
Erlotinib treatment of EGFR-mutant cells was compared to MEK inhibitor treatment of BRAF-mutant melanoma cells lines and CL-387,785 (irreversible EGFR inhibitor) treatment of EGFR-mutant lung cancer …
The data for the 24 hr time points are the same as in Figure 2A. p-values are shown for the comparison of mean SOX2 fluorescence of each treated population to DMSO (Student's t-test, unequal …
(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 …
Raw immunofluorescence data for quantitation of SOX2 staining in HCC827 cells with erlotinib treatment in Figure 2A, and SOX2+ Ki67 staining in Figure 2—figure supplement 3.
Raw immunofluorescence data for quantitation of SOX2 staining in PC9 cells with erlotinib treatment in Figure 2A and Figure 1—figure supplement 4.
Raw immunofluorescence data for quantitation of SOX2 staining in HCC827 cells recovered after retreatment (x2) with erlotinib, compared to previously untreated, in Figure 2B.
Raw immunofluorescence data for quantitation of SOX2 staining in HCC827 and PC9 cells with increasing dose of erlotinib in Figure 2—figure supplement 1.
Raw immunofluorescence data for quantitation of SOX2 staining in PC9 cells recovered after retreatment (x2) with erlotinib, compared to previously untreated cells, in Figure 2—figure supplement 4A.
Raw immunofluorescence data for quantitation of phospho-EGFR (pY1068) in parental and erlotinib-resistant PC9 cells in Figure 2—figure supplement 4B.
HCC827 (left) and PC9 (right) cells were treated for 24 hr with the indicated dose of erlotinib, followed by immunofluorescence microscopy with antibodies to SOX2 and DAPI. The distribution of SOX2+ …
Immunofluorescence microscopy was carried out on erlotinib-treated HCC827 (upper panels) or PC9 (lower panels) cells using antibodies to SOX2 and various stem cell markers. CD133 (upper row of …
Left panels, HCC827 cells were treated for 24 hr with 0.1 µM erlotinib, followed by immunofluorescence microscopy with antibodies to SOX2, Ki-67, and DAPI. The left three pairs of panels show HCC827 …
(A) Retreatment of PC9 cells after a period of recovery does not increase the fraction of cells capable of inducing SOX2. Left panels, images of cells stained for SOX2 (green) and DAPI (blue). Right …
(A) Immunofluorescence analysis of colonies formed from single HCC827 cells for DAPI (blue), SOX2 (green), and Ki67 (red). (B) Similar analysis of DAPI (blue) and SOX2 (green) in PC9 cells. For …
Data are shown as mean Ct (normalized to GAPDH and untreated cells) of 3 replicates −/+ SEM.
Nude mice were xenografted subcutaneously with PC9 cells and treated with a single oral dose of erlotinib (100 mg/kg) (or carrier) when the tumors had reached ∼500 mm3. Tumors were harvested 24 hr …
Number of SOX2+cells per field for quantitation of SOX2 staining in PC9 cell xenografts in Figure 3.
Raw absorbance data for quantitation of SOX2 staining in HCC827 cell xenografts in Figure 3—figure supplement 1.
Quantitative immunohistochemistry for SOX2 was performed in FFPE sections from mice xenografted subcutaneously with HCC827 cells and treated with a single oral dose of erlotinib (red in dot plot) or …
Short-term cultures of tumor cells derived from patients at the time of acquired resistance (both tumors EGFR genotype exon 19 deletion + T790M) were treated with the indicated agents for 24 hr, …
Raw immunofluorescence data for quantitation of SOX2 staining with different treatments in patient-derived tumor cells.
(A) HCC827 cells were stably transduced with a doxycycline inducible epitope-tagged SOX2 lentiviral expression vector. Doxycycline was added for 3 hr (‘SOX2-tag’) and then removed prior to the …
Raw immunofluorescence data for quantitation of SOX2 staining in HCC827 cells with inducible SOX2 in Figure 5—figure supplement 1A.
Raw immunofluorescence data for quantitation of SOX2 and cleaved caspase-3 costaining in PC9 cells transfected with siCTRL or siSOX2 in Figure 5—figure supplement 2.
(A) Quantitative immunofluorescence analysis confirms that ectopic SOX2 expression with a short pulse of doxycycline increases the fraction of SOX2+ cells without significantly increasing the amount …
Left panel, quantitative immunofluorescence analysis showing expression of SOX2 (x-axis) and cleaved caspase-3 (y-axis) in PC9 cells transfected with siCTRL (blue) or siSOX2 (red) and treated with …
(A), PC9 cells were transfected with two different siRNA duplexes targeting SOX2 (or control siRNA), followed by addition of DMSO or 0.1 µM erlotinib for 24 hr and immunoblot of protein lysates with …
Each BIM isoform (EL, L, and S) was normalized to the GAPDH loading control and untreated, siCTRL cells.
(A) The levels of transcripts for each of the indicated BH3 domain-containing proteins was assessed by quantitative PCR at multiple time points after erlotinib treatment in uninduced PC9 cells (blue …
Induction of BIM, BMF, and HRK following erlotinib treatment of HCC827 cells was assessed as in Figure 6A.
(A) Putative FOXO protein binding sites within the promoter of SOX2, identified using TRANSFAC and Zhang et al., 2011. (B) HCC827 cells were transfected with control siRNA or siRNA targeting the …
Raw immunofluorescence data for quantitation of SOX2 staining with different FOXO protein knockdown in Figure 7C.
Raw immunofluorescence data for quantitation of SOX2 and FOXO6 costaining in HCC827 cells in Figure 7—figure supplement 3.
(A) MSigDB/TRANSFAC output for the 12 genes most highly upregulated by erlotinib (FDR <0.05). (B) Although binding sites for FOXF2 are also enriched, knockdown of FOXF2 does not decrease …
(A) Same lysates as Figure 7E, showing immunoblot for FOXO proteins. (B) Similar data as in Figure 7B, but shown after immunoblot of protein lysates with the indicated antibodies. Immunoblot for …
(A) HCC827 cells were left untreated or were treated with 0.1 µM erlotinib for 24 hr. Cells were stained with goat anti-SOX2 and rabbit anti-FOXO6 primary antibodies, followed by anti-goat-Alexa …
(A) Pre-treatment of PC9 cells with FGF10 has minimal effects on SOX2 induction by erlotinib. (B) The addition of exogenous Wnt3A has no effect on induction of SOX2 by erlotinib. (C) The …
(A) HCC2935 cells were transfected with siCTRL or siSOX2 48 hr prior to erlotinib addition and assayed for cytoxicity 48 hr later with Syto-60. Data are displayed as the mean of 5 replicates −/+ …
Raw immunofluorescence data for quantitation of SOX2 staining in HCC2935 cells in Figure 8B.
In untreated cells, mutant EGFR drives cell survival by activating downstream signaling pathways, including PI3K and MAPK, which inhibit apoptosis through transcriptional and post-transcriptional …
siRNA, primer, and probe sequences/sources used in the study.