Leucine-rich repeat-containing G protein-coupled receptor 5 marks different cancer stem cell compartments in human Caco-2 and LoVo colon cancer lines

Figure 1 Proliferation and differentiation analysis of the stem cell compartment in stably transfected clones. A and B: Phase contrast and fluorescence images show colonies of stably transfected Caco-2 (A) and LoVo (B) cells expressing the enhanced yellow fluorescent protein (EYFP) reporter (red arrows show small cells expressing high level of enhanced yellow fluorescent protein, EYFP); C and E: Fluorescence-activated cell sorting (FACS) sorting of leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5)/EYFP expression in Caco-2 clone (C) and LoVo clones (E) into EYFP^high, EYFP^low, and EYFP^negative populations; D and F: Quantitative real-time polymerase chain reaction analysis of LGR5 messenger ribonucleic acid expression in EYFP^high, EYFP^low, and EYFP^negative cells relative to glyceraldehyde-3-phosphate dehydrogenase gene in Caco-2 clones (D) and LoVo clones (F); G: Phase contrast images show proliferation of EYFP^high cells from LGR5/EYFP Caco-2 clone; fluorescence images show self-renewal of LGR5/EYFP stem cells and down-regulation of LGR5/EYFP in differentiated progeny in LGR5/EYFP Caco-2. Sequential FACS analysis shows EYFP expression in LGR5/EYFP Caco-2 clone over 6 d in culture; H: Phase contrast images show proliferation of EYFP^negative and cells from LGR5/EYFP Caco-2 clone; fluorescence images show self-renewal of LGR5 stem cells and down-regulation of LGR5 in differentiated progeny in LGR5/EYFP Caco-2. Sequential FACS analysis shows EYFP expression in LGR5/EYFP Caco-2 clone over 6 d in culture; I and J: Phase contrast images show proliferation of EYFP^high (I) and EYFP^negative (J) cells from LGR5/EYFP LoVo clone; fluorescent pictures showing self-renewal of LGR5 stem cells and down-regulation of LGR5/EYFP in differentiated progeny in LGR5/EYFP LoVo; sequential FACS analysis of EYFP expression in LGR5/EYFP LoVo clone over 6 d in culture. LGR5: Leucine-rich repeat-containing G protein-coupled receptor 5; EYFP: Enhanced yellow fluorescent protein.