Fig. 2: CtBP2 confers metabolite-sensing capabilities to FoxO1.
a The NADH-sensing capability of CtBP2/FoxO1 complex. Increasing concentrations of NADH (0, 0.5, 5, 50 μM) were added to the HEK293 cell lysates expressing indicated proteins. b Direct binding of recombinant CtBP2 protein to oleoyl-CoA and NADH analyzed by MST (n = 3 or 4 for each point). c Computer-assisted structural analysis of CtBP2 with palmitoyl-CoA. Palmitoyl-CoA is localized at the interface of CtBP2 adopting dimeric configuration. d The fatty acyl-CoA-sensing capability of CtBP2/FoxO1 complex. Increasing concentrations of oleoyl-CoA (0, 50, 150, 500 μM) were added to the HEK293 lysates. e The thermal shift induced by NADH and oleoyl-CoA binding in DSF. Recombinant CtBP2 were mixed with either NADH (20 μM) or oleoyl-CoA (20 μM) and thermal stability was monitored on a temperature gradient. f The effect of cytosolic redox state on CtBP2/FoxO1 complex. HEK293 cells expressing FLAG-FoxO1 and CtBP2 were incubated with different ratios of lactate and pyruvate for 1 h. g HEK293 cells expressing indicated proteins were incubated with control BSA or palmitate-BSA conjugate (200 μM) for 30 min. h Schematic diagram showing the split luciferase complementation assay. i HEK293 cells stably expressing the reporter were stimulated with 50 mM lactate. The arrow indicates the addition of either vehicle or lactate (n = 4, biologically independent cells). The p values are as follows: 1.3 × 10−3 for 1 min, 2.8 × 10−3 for 3 min, 1.8 × 10−3 for 5 min, 6.0 × 10−4 for 10 min, 7.3 × 10−4 for 15 min, 5.6 × 10−4 for 20 min, 6.5 × 10−4 for 25 min, 6.2 × 10−4 for 30 min. Data are expressed as the mean ± SEM. ** denotes p < 0.01 evaluated by unpaired two-tailed Student’s t-test. Source Data are provided as a Source Data file.
