Figure 1.
SP analysis in cultured H460 lung cancer cells.
A–B, Hoechst staining of H460 cells. Note that although the majority of cells were stained in the nucleus, some cells (indicated by arrows) apparently lacked nuclear Hoechst staining. C–D, SP phenotypes in the absence (C) or presence (D) of verapamil.
Figure 2.
Sphere formation and proliferative capacity of H460 SP and non-SP cells.
A–B, Purified SP and non-SP cells were cultured in serum-free medium in anchorage-independent conditions. The SP cells formed typical floating spheres within 4 days (A) whereas the non-SP cells largely established adherent growth (B). C, The SP cells displayed higher proliferative ability than non-SP cells as determined by CCK-8 kit (P<0.05 for all time points, Student t-test).
Figure 3.
SP re-analysis in various samples.
A–B, SP re-analysis when the SP spheres were disaggregated and dissociated cells cultured in normal serum-containing medium for one week. C–D, SP re-analysis in the SP cell-derived tumors. E–F, SP re-analysis in non-SP cell-derived tumors. A, C, E, without verapamil. B, D, F, with verapamil.
Figure 4.
High tumorigenicity in SP cells.
A, Table presentation of the tumorigenic potential of H460 SP and non-SP cells. Three parameters of tumorigenicity, i.e., tumor incidence, latency, and volume (*P<0.01) were shown. All animals were terminated (term.) 28 days after implantation. B, The SP cells regenerated larger tumors than corresponding non-SP cells at every cell dose. C, Gross tumor images when tumors were harvested at day 28 after s.c. injection of the SP and non-SP cells into NOD/SCID mice. D, Representative HE-stained photomicrographs of SP and non-SP tumors.
Figure 5.
RT-PCR analysis of ABCG2 and SMO mRNA levels.
A, Representative RT-PCR gel images. M, marker; lane 1, NCI-H460 cells normally cultured in serum-containing medium; lane 2, the H460 spheres; lane 3, purified SP cells; lane 4, purified non-SP cells; lane 5, the Tomatidine control group; lane 6, the Cyclopamine experimental group; lane 7, SP tumors; lane 8, non-SP tumors. B, Quantitative presentation of ABCG2 and SMO mRNA levels as determined by densitometry (P<0.001, except ABCG2 mRNA lane 6 vs. lane 8 and SMO mRNA lane 1 vs. lane 5 and lane 4 vs. lane 6).
Figure 6.
Cyclopamine inhibits H460 cell proliferation.
A–C, Representative photomicrographs of H460 cells 72 h after treatment with vehicle control (A), Tomatidine (B), or Cyclopamine (C). Original maginifications: ×200. D, Cyclopamine dose-dependently inhibited H460 cell proliferation (P<0.05; one-way ANOVA). E, Time course of Cyclopamine inhibition of H460 cells (P<0.05, one-way ANOVA). Cyclopamine was used at 20 µmol/L. F, Effects of Cyclopamine on the cell cycle of H460 cells.