H1650 and H1650-ER1 cells were cultured under non-adherent conditions in serum free medium and spheroids were counted after 15 days. (A) Images of spheroids generated by H1650 and H1650-ER1 cells. The scale bar corresponds to 50 μm. (B) Quantification of spheroids generated. H1650 and H1650-ER1 cells were seeded in 6 well plates at a density of 6000 cells per well. The resistant subline generated a significantly higher number of spheroids (p-value < 0.01). Each data point represents the mean of three independent experiments. The error bars represent s.e.m. (n = 3). (C) Clonogenicity of H1650-ER1 cells determined by limiting dilution assay. Cells were seeded in 6 well plates at density varying from 12-6000 cells per well. Each data point represents the mean of 6 wells. The error bars represent S.D. (n = 6). (D) Self renewal ability was determined by dissociating spheres into single cells, replating them under non adherent conditions, and counting generation of secondary spheres after 15 days. Each data point represents the mean of 6 replicates. The error bars represent S.D. (E) mRNA expression of OCT3/4, NANOG, BMI-1 and STAT3 in H1650-ER1, ER1 spheroids (3rd generation) and adherent cells were measured by quantitative RT-PCR. Fold change expression was normalized with respect to H1650-ER1 cells. The error bars represent s.e.m. (n = 3).