Fig. 5: Deletion of ALDOA leads to imbalanced glycolysis.
a, Diagram depicting the autocatalytic structure of glycolysis and allosteric regulation of PFK1. Figure ceated in BioRender (BioRender.com). b, FBP levels in control (shRen) or ALDOA-depleted (shAldoA.1280) Akt1myr;MycOE;Trp53–/– cells after 48 h doxycycline treatment (1 µg ml−1) followed by addition of fresh medium including doxycycline for indicated times. Data are presented as mean ± s.d. (n = 3 biologically independent replicates). c, ATP levels in control (shRen) or ALDOA-depleted (shAldoa.1280, shAldoa.558) cells after 48 h of doxycycline treatment (1 µg ml−1) followed by addition of fresh medium for 6 h. Data are presented as mean ± s.d. Significance was calculated using a two-tailed Student’s t-test with FDR correction (n = 3 biologically independent replicates). d, Dynamic mathematical model of glycolysis simulating levels of FBP, ATP and Pi in controls (ALDOA 100% glucose 5 mM, left), ALDOA-depleted (ALDOA 20% glucose 5 mM, middle) or ALDOA-depleted cells after glucose deprivation (ALDOA 20% glucose 1 mM, right). e, Pi in control (shRen) or ALDOA-depleted (shAldoa.1280, shAldoa.1235) cells treated as in a. Data are presented as mean ± s.d. Significance was calculated using one-way ANOVA with Dunnett’s post hoc test (n = 6 independent biological replicates). f, Pi in control (sgControl) or Gpi knockout (sgGpi #1/#4, sgGpi #4 cl.1) cells after addition of fresh medium for 6 h. Data are presented as mean ± s.d. Significance was calculated using one-way ANOVA with Dunnett’s post hoc test (n = 3 biologically independent replicates). g, Bifurcation analysis. Diagram shows the parameter regions corresponding to stable or unstable states as a function of Vmax, ALD and [GLCe]. Maximal ALDOA activity is expressed as a percentage of reference value. h, Levels of FBP and Pi as well as the ATP:ADP ratio in control (shRen) and ALDOA-depleted (shAldoa.558 and shAldoa.1280) cells after incubation in fresh MPLM medium containing 4.4 mM or 0.44 mM glucose for 6 h. Data are presented as mean ± s.d. Significance was calculated using one-way ANOVA with Dunnett’s post hoc test (biologically independent replicates: n = 5 shAldoa.558 4.4 mM and shAldoa.1280 0.44 mM, n = 4 others). i, Activation of AMPK determined by phospho-ACC (S79) in control (shRen) and ALDOA-depleted (shAldoa.1280) cells after culture in medium containing indicated glucose concentrations for 6 h. Vinculin is shown as loading control. j, Proliferation of control (shRen) or ALDOA-depleted (shAldoa.1280) cells in medium containing indicated glucose concentrations. Data are presented as mean ± s.d. (n = 3 biologically independent replicates).
