The ALDEFLUOR assay kit was used to identify the stem and progenitor inhibitor bulk cell populations according to manufacturers instructions. BODIPY ami noacetaldehyde was used as a substrate, and diethylaminobenzaldehyde was used as an inhibitor for negative controls. Cell surface bound EGFR was mea sured using a phycoerythrin conjugated EGFR anti body and PE conjugated mouse IgG2b isotype control antibody. Following gentle cell dissociation or ALDEFLUOR assay, the cells were washed, resuspended in 80 ul of PBS with bovine serum Inhibitors,Modulators,Libraries albumin or ALDEFLUOR assay buffer and 20 ul of either antibody or isotype control solution were added. Reactions were incubated on ice for 30 minutes, the cells were washed with either PBS and BSA or ALDEFLUOR assay buffer and resuspended in 0. 5 ml of PBS or ALDEFLUOR assay buffer.

QuantiBrite beads were used to estimate the number of EGFR molecules per Inhibitors,Modulators,Libraries cell. Samples were measured using a FACSAria II Cell Sorter 5 laser SORP instrument or sorted using a MoFlo sorter. Immunofluorescence Cells cultured on coverslips for 24 hours were fixed for 10 minutes at room temperature in 3% paraformalde hyde 2% sucrose Inhibitors,Modulators,Libraries solution, rinsed twice with PBS and per meabilized with ice cold Triton X 100 solution 1 piperazineethanesulfonic acid pH 7. 4, 50 mM NaCl, 3 mM MgCl2, 300 mM sucrose for 3 minutes on ice. The cells were rinsed for 5 times with PBS and blocked for 20 minutes with 10% goat serum followed by incubation with primary antibody anti EGFR and anti ALDH1A1 for 20 minutes at 37 C.

Cells were Inhibitors,Modulators,Libraries washed two times and incubated for 20 minutes at 37 C with secondary antibody Alexa 488 conjugated anti rabbit or Alexa 594 conjugated anti mouse antibody. The nuclei were stained with DAPI, and the slides were examined using a Nikon fluorescence microscope. For quantification of the fluorescence signal, the mean inten sity was determined using ImageJ software in four Inhibitors,Modulators,Libraries differ ent fields for each sample. Experiments were performed in triplicate, and the means and standard deviations of the signal intensities were calculated for each condition. Real time RT PCR Total RNA was extracted using the RNeasy Plus Mini Kit. RNA was reverse transcribed using the AccuScript enzyme in the AccuScript High Fidelity RT PCR System. A quantitative real time RT PCR assay was carried out on a Rotor Gene 6000 cycler using SYBR Green Supermix.

The PCR reaction was per formed under the following conditions, 95 C for 10 min utes followed by 45 cycles at 95 check details C for 20 seconds, at 56 C for 25 seconds and at 72 C for 40 seconds. The expression of the EGFR gene was normalized to GAPDH Immunohistochemistry, morphometry and statistics Immunohistochemistry was performed as described pre viously. Scoring for EGFR expression was done according to the following system, Score 0 no staining or staining in less than 10% of cells. Score 1, a faint perceptible membrane staining can be detected in more than 10% of cells.