Extended Data Fig. 4: H2O2 is generated upon D-alanine treatment in a dose-dependent fashion in DAAO-expressing tumour cells and lung fibroblasts.
From: Unchecked oxidative stress in skeletal muscle prevents outgrowth of disseminated tumour cells
a) Workflow to test if D-/L-alanine stimulated H2O2 in MDA231-WT and -DAAO. b) Extracellular H2O2 of MDA231-WT and -DAAO treated with D-/L-alanine. n = 3 replicates, performed twice. Two-way ANOVA was used, followed by Dunnett’s test. Compared against untreated MDA231-DAAO mean- 1 h: *P = 0.028,100 mM D-ala. 4 h: ***P = 0.0001, 100 mM D-ala; **P = 0.0017, 20 mM D-ala; **P = 0.0019, 100 mM D-ala MDA231-WT. 8 h: ***P = 0.0001, 100 mM L-ala, 20 mM L-ala. *P = 0.023, 100 mM D-ala MDA231-WT; * P = 0.018, 20 mM D-ala MDA231-WT. 24 h: ***P = 0.000, 100 mM D-ala; *P = 0.011, 100 mM L-ala. 48 h: ***P = 0.000, 100 mM D-ala. All other P > 0.05. c) MDA231-WT and -DAAO outgrowth with D-/L-alanine. n = 3 replicates, performed twice. Two-way ANOVA, followed by Dunnett’s test: compared against untreated MDA231-DAAO- 48 h: ***P = 0.0001, 100 mM D-ala; **P = 0.0011, 100 mM D-ala MDA231-WT; **P = 0.0036, 20 mM D-ala MDA231-WT; *P = 0.04, untreated MDA231-WT. 72 h: ***P = 0.0001, 100 mM D-ala, untreated MDA231-WT, 20 mM L-ala MDA231-WT. All others P > 0.05. d) Workflow to test if D-/L-alanine stimulates H2O2 in LF-DAAO. e) Extracellular H2O2 in LF-DAAO and LF treated with D-/L-alanine. n = 3 replicates per condition, performed twice. Two-way ANOVA, followed by uncorrected Fisher’s LSD: ****P < 0.0001, LF-DAAO 100 mM D-ala v. LF 100 mM D-ala, LF-DAAO 100 mM D-ala v. LF-DAAO 100 mM L-ala (4 h). 8 h: *P = 0.03, LF-DAAO 50 mM D-ala v. LF 50 mM D-ala; ****P < 0.0001, 70-, 100-mM D-ala LF-DAAO v. LF. 24 h: * P = 0.033, LF-DAAO 20 mM D-ala v. LF 20 mM D-ala; ** P = 0.004, LF-DAAO 30 mM D-ala v. LF 30 mM D-ala. **** P < 0.0001, 40-, 50-, 70-, 100-mM D-ala LF-DAAO v. LF. 48 h: *** P = 0.0006, LF-DAAO 30 mM D-ala v. LF 30 mM D-ala. **** P < 0.0001, 40-, 50-, 70-, 100-mM D-ala LF-DAAO v. LF. Other LF-DAAO v. LF comparisons with D-ala P > 0.05. For b-c and e, error bars represent the s.e.m.
