Decreased <i>in vivo</i> tumorigenic activity of FACS-purified CD44+ and CD44−/CD133− subpopulations from control or HIF-1α or HIF-2α-knockdown SW480 cells.

<p><b>A)</b> Analysis of CD44/CD133 expression profiles in the SW480 colorectal cancer cell line. The cells were incubated in the presence of EDTA and washed, and 1×10⁵ cells were incubated with PE-conjugated anti-CD133 and allophycocyanin-conjugated anti-CD44 antibodies for 15 min at 4°C and examined using flow cytometry. <b>B)</b> The CD44⁺ (population corresponding to the upper left quadrant in panel A) or CD44⁻/CD133⁻ (population corresponding to the lower left quadrant) subpopulations from the stable control or HIF-1α- or HIF-2α-silenced cells were obtained by FACS cell sorting. The purified subpopulations were s.c. injected into the dorsal region of healthy six-week-old immunocompromised nude mice (n = 5 for each condition). Each mouse was injected in the right flank with the CD44⁻ cells and in the left flank with the CD44⁺ cells. A total of 1×10⁴ cells in 100 µl of growth factor- reduced Matrigel were transplanted at each site. Four weeks after inoculation, the animals were euthanized, and the tumors were removed and weighed. The data represent the means ± SEM for each experimental condition with n = 5. **: p<0.05; ***: p<0.01.