Fig 1.
Bioenergetic analyses of CSCs and non-stem cancer cells in SCLC cell line H446.
(A) Basal OCR and ECAR for uPAR+ and uPAR- subsets. (B) The OCR after treatment with indicated inhibitors in uPAR+ cells versus uPAR- cells. (C) Maximum mitochondrial respiratory capacities and mitochondrial reserve capacities in two subpopulations. (D) The ECAR after treatment with indicated inhibitors in uPAR+ cells versus uPAR- cells. (E) The compensative potential of glycolysis of uPAR+ cells and uPAR- cells. (F) ATP content in uPAR+ cells and uPAR- cells. Data are expressed as mean ±s.e.m. of three independent experiments. Student’s t-test was used to calculate statistical significance. *P<0.05.
Fig 2.
Glucose uptake and lactate production in CSCs and non-stem cancer cells.
(A) Glucose uptake by uPAR+ cells and uPAR- cells using 2-NBDG. Left, histogram representation of 2-NBDG intensity. Right, quantification of 2-NBDG uptake as difference in mean fluorescence intensity (MFI) between samples and controls. (B) Lactate production rates of uPAR+ cells and uPAR- cells. Data are expressed as mean ±s.e.m. of three independent experiments. Student’s t-test was used to calculate statistical significance. *P<0.05.
Fig 3.
Numbers and morphology of mitochondria in CSCs and non-stem cancer cells.
Ultrastructural analysis of sorted uPAR+ cells and uPAR- cells using transmission electron microscopy. Representative images are shown at magnification of ×10,000 in upper panels and ×30,000 in lower panels. Arrows in lower panel indicate examples of mitochondria. Scale bars: 5 μm (upper panels) and 1 μm (lower panels).
Fig 4.
Effect of metabolic inhibitors on ATP levels in CSCs and non-stem cancer cells.
(A, B) ATP content of uPAR+ cells and uPAR- cells treated with 2-DG, oligomycin (OLI) or a combination of 2-DG and OLI. (C) The effect of combination of 2-DG and OLI for various time on intracellular ATP levels in uPAR+ cells and uPAR- cells. (D) The uPAR+ cells, not the uPAR- cells could produce more ATP through mitochondrial substrate-level phosphorylation under hypoxic conditions than normoxic conditions (E) The addition of substrates (SUB) could remedy the ATP depletion caused by 2-DG and OLI in uPAR+ cells but not in uPAR- cells. Data are expressed as mean±s.e.m. of three independent experiments. Student’s t-test was used to calculate statistical significance. *P<0.05. SUB, substrates (α-ketoglutarate and aspartate). ns, no significance.
Fig 5.
Impairment of oligomycin on sphere-forming and proliferative abilities of CSCs in H446 cells.
(A) Representative morphology of spheres maintaining in serum-free medium were established from uPAR+ cells treated with oligomycin and 2-DG. (B) Amount of the first-generation spheres generated from uPAR+ cells treated with 2-DG and oligomycin. Clonogenic potential of uPAR+ cells was reduced by oligomycin treatment. In contrast, 2-DG does not significantly affect the clonogenic potential of CSCs. (C) Number of cells per sphere generated from uPAR+ cells treated with 2-DG and oligomycin on the day of 14. (D) Oligomycin inhibits proliferation of both the uPAR+ cells and uPAR- cells, however 2-DG affects only uPAR- cells but not uPAR+ cells. Data are expressed as mean ± s.e.m. of three independent experiments. Student’s t-test was used to calculate statistical significance. *P<0.05.
Fig 6.
The effect of oligomycin on CSCs tumor formation ability in vivo.
(A) Experimental scheme. (B) Representative photograph of tumors formed by the cells treated with oligomycin or control cells. (C) Representative photograph of BALB/cA nude male mice with tumor at 42 days. (D) Tumor weight of xenograft tumors volume of the group treated by oligomycin and control group. (E) Growth curve of xenograft tumors volume of the group treated by oligomycin and control group. Data are expressed as mean ± s.e.m. of six mice. Student’s t-test was used to calculate statistical significance. *P<0.05.