Fig. 3: Cell adhesion and cell area are controlled by the glycolytic metabolite FBP.
From: Fructose-1,6-bisphosphate couples glycolytic activity to cell adhesion
a, Loss of aldolase results in severely increased FBP levels, whereas PFK depletion lowers FBP. Relative FBP levels measured in U-2 OS cells treated with indicated siRNAs. Data were normalized to siCtrl and represent mean ± s.e.m.; n = 3 independent experiments; two-sided one-sample t-test (P values: (siPFK) = 0.0345; (siALDOA) = 0.0425; (siGAPDH) = 0.2188). b, Efficient codepletion of PFK and aldolase. Immunoblot of U-2 OS cells treated with indicated siRNAs. Clathrin heavy chain (CHC) was used as loading control; N = 1 independent experiment. c, Codepletion of PFK restores normal FBP levels in aldolase-knockdown cells. Relative FBP levels measured in U-2 OS cells treated with indicated siRNAs. Data were normalized to siCtrl and represent mean ± s.e.m.; n = 4 independent experiments; two-sided one-sample t-test (P values: (siCtrl + siPFK) = 0.0017; (siCtrl + siALDOA) = 0.0491; (siPFK + siALDOA) = 0.16). d–f, Co-depletion of PFK restores FA numbers and cell size in aldolase knockdown cells. d, Representative confocal images of paxillin-labelled FAs in U-2 OS cells treated with indicated siRNAs. FA segmentation and cell outlines (red) are shown on the right. e,f, A quantification of FAs per cell (e) and cell area (f) shown in d. For e,f, data represent mean ± s.e.m.; n = 5 independent experiments; one-way ANOVA with Dunnett’s post-test (P values (for e): (siCtrl + siCtrl versus siCtrl + siPFK) = 0.0015; (siCtrl + siCtrl versus siCtrl + siALDOA) = 0.0002; (siCtrl + siCtrl versus siPFK + siALDOA) = 0.1021; P values (for f): (siCtrl + siCtrl versus siCtrl + siPFK) = 0.0044; (siCtrl + siCtrl versus siCtrl + siALDOA) <0.0001; (siCtrl + siCtrl versus siPFK + siALDOA) = 0.8423). g,h, Inhibiting glycolytic flux rescues FAs per cell (g) and cell area (h) in aldolase-knockdown cells. A quantification of FAs per cell and cell area of U-2 OS cells treated with control (−) or ALDOA-specific siRNA (+) followed by 48 h treatment with PBS (−) or 25 mM 2-DG (+). Data represent mean ± s.e.m.; n = 5 independent experiments; one-way ANOVA with Dunnett’s post-test (P values (for g): (siCtrl + PBS versus siALDOA + PBS) = 0.0056; (siCtrl + PBS versus siCtrl + 2-DG) = 0.0567; (siCtrl + PBS versus siALDOA + 2-DG) = 0.1605; P values (for h): (siCtrl+PBS versus siALDOA+PBS) <0.0001; (siCtrl + PBS versus siCtrl + 2-DG) = 0.2411; (siCtrl + PBS versus siALDOA + 2-DG) = 0.9888). Corresponding images are shown in Extended Data Fig. 5a. n.s., not significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bars, 25 µm.
