Fig. 7: Glycolytic waves regulate the energy shift in cancer.

a Left: Quantification of wave activity from previous published work¹⁴ is re-plotted for comparison (mean ± SD of fraction of cells with waves during a 2 h imaging window, 501 M1 cells, 608 M2 cells, 234 M3 cells, and 302 M4 cells from 4 independent experiments). Welch’s ANOVA test was performed for the four groups, ****p < 0.0001. Right: Quantification of intracellular iATP cpGFP/mRuby ratio, normalized to the mean of M1 cell, in M1-M4 MCF-10A cells pretreated in OAR (violin plot and mean ± SD of 112 M1 cells, 139 M2 cells, 200 M3 cells, and 198 M4 cells from 5 independent experiments). Welch’s ANOVA test was performed for the four groups, ****p < 0.0001. b Normalized iATP cpGFP/mRuby ratio signal shown in mean ± SEM of n = 27 M1 cells (green) and n = 38 M3 cells (red) from 4 independent experiments treated with LY294002 at the indicated time. The cpGFP/mRuby values are normalized to the time point that LY294002 was added. Cells were pretreated in OAR before the treatment with LY294002. c Fraction of iATP cpGFP/mRuby drop upon treatment with LY294002 in these 27 M1 cells (green) and 38 M3 cells (red) in (b) were shown as violin plots (with quartiles and median) and compared by two-tailed unpaired t test with Welch’s correction, ***p = 0.0007. d Left: Quantification of wave activities in 7 cancer cell lines (mean ± SD of fraction of cells with waves during a 2 h imaging window; 289 MCF-7 cells, 208 Calu-6 cells, 108 SNU-387 cells, 221 HepG2 cells, 301 HCT116 cells, 275 MDA-MB-231 cells, and 220 AsPc-1 cells from 4 independent experiments except SNU-387 cells from 8 independent experiments are quantified). Welch’s ANOVA test was performed for the 7 groups, ****p < 0.0001. Representative confocal images showing the wave activities in these 7 cell lines are displayed in Supplementary Fig. 12 and Supplementary Movie 17. Right: Quantification of iATP cpGFP/mRuby ratio, normalized to the mean of MCF-7 cell, in 7 cancer cell lines pretreated in OAR (violin plot and mean ± SD of 175 MCF-7 cells, 177 Calu-6 cells, 110 SNU-387 cells, 124 HepG2 cells, 163 HCT116 cells, 140 MDA-MB-231 cells, and 105 AsPc-1 cells from at least 4 independent experiments). Welch’s ANOVA test was performed for the 7 groups, ****p < 0.0001. In both plots, from left to right, the cell lines are: MCF-7, Calu-6, SNU-387, HepG2, HCT116, MDA-MB-231, and AsPc-1, each represented by a unique color. e Quantification of fraction of iATP cpGFP/mRuby drop upon treatment with DB (left panel) or OAR (right panel) in 7 cancer cell lines (violin plot and mean ± SD of 45 DB-treated and 35 OAR-treated MCF-7 cells, 38 DB and 33 OAR Calu-6 cells, 27 DB and 25 OAR SNU-387 cells, 37 DB and 22 OAR HepG2 cells, 38 DB and 31 OAR HCT116 cells, 38 DB and 31 OAR MDA-MB-231 cells, and 37 DB and 38 OAR AsPc-1 cells from at least 4 independent experiments). Welch’s ANOVA was performed for the seven cell lines upon the treatment with DB (left) or OAR (right), respectively; ****p < 0.0001. In both plots, from left to right, the cell lines are: MCF-7, Calu-6, SNU-387, HepG2, HCT116, MDA-MB-231, and AsPc-1, each represented by a unique color. f–h The mean ± SEM of intracellular ATP levels in cells pretreated with OAR as shown in (d), and the mean ± SEM of ATP reduction fractions in cells upon treatment of DB or OAR as shown in (e), are plotted against the mean ± SEM of wave activities among the 7 cancer cell lines from (d), resulting in the new graphs (f), (g,h), respectively. Linear regression trend lines and corresponding R² values are shown in each plot. Cell lines from left to right in all three plots are MCF-7, Calu-6, SNU-387, HepG2, HCT116, MDA-MB-231, and AsPc-1, each indicated by a unique color.