Fig. 3: Increased mitochondrial 8-oxo-dG accumulation in thrombin-stimulated MTH1-deficient platelets.

a Accumulation of 8-oxo-dG in mitochondria of platelets from MTH1^fl/fl or MTH1^−/− mice after stimulation by thrombin (1 U/ml) or CRP (5 μg/ml) (mean ± SE, n = 3 independent isolated platelets, two-way ANOVA with Sidak multiple comparisons test). b Washed platelets were loaded with MitoSOX Red (5 μM) for 10 min and then stimulated with thrombin (0.25 U/ml) for 3 min to measure mitochondrial ROS production by flow cytometry (mean ± SE, n = 3 independent isolated platelets, two-way ANOVA with Sidak multiple comparisons test). c PAR3 and PAR4 expression in MTH1^fl/fl and MTH1^−/− platelets under resting conditions (mean ± SE, n = 3 independent isolated platelets, two-tailed unpaired Student’s t test). d Wild-type (WT) platelets were treated with thrombin (1 U/ml) or CRP (5 μg/ml) for 3 min followed by measuring the mitochondrial ROS generation using MitoSox Red and MitoTracker Green probes by fluorescent microscope (×100) (n = 3 independent isolated platelets). e Representative image of mitochondrial ROS generation in WT platelets after stimulation with CRP or thrombin by flow cytometry. f WT platelets were pretreated with BAPTA (calcium inhibitor) (20 μM), BAY 11-7082 (NF-κB inhibitor), U-73122 (PLC inhibitor) (5 μM), LY294002 (PI3K inhibitor) (20 μM), PP1 (Src inhibitor) (10 μM) (MedChemExpress) for 5 min followed by stimulation with thrombin or CRP to measure mitochondrial ROS by flow cytometry (mean ± SE, n = 3 independent isolated platelets, one-way ANOVA with Dunnett multiple comparisons test). g WT platelets were pre-incubated with vehicle, Mito-TEMPO (10 μM) or Apocynin (500 μM) and then treated with thrombin or CRP followed by measuring intracellular ROS production by flow cytometry using H2DCFDA (mean ± SE, n = 3 independent isolated platelets, two-way ANOVA with Tukey multiple comparisons test).