Cytokine-withdrawn cells after initial IL-1β/TGF-β exposure retain stem-like properties. (A) Representative images of LN-229 cells cultured in SFM in the absence (CTL, (control)) or presence of IL-1β/TGF-β (CT, (IL-1β/TGF-β)), or after withdrawal of both cytokines following initial exposure (CW, (IL-1β/TGF-β → CTL)) for seven days. Scale bar = 200 μm. (B) qRT-PCR analysis of gene expression of stem cell markers in CTL, CT and CW cells. β-actin was used as an internal normalization control. (C) The number and size of colonies formed in soft agar. CTL, CT and CW cells were dissociated and 10,000 cells/well of each population were cultured in soft agar in six-well plates for 21 days. Colony number was counted under a microscope and colony size was measured using ImageJ. Twenty random fields were selected for the measurement and the data represent the average colony number and size per microscope field. *P < 0.05. **P < 0.01. Error bars represent SEM.