Figure 7.
CircSamd4 decreases mitochondria-derived ROS by recruiting Vcp to the mitochondria
(A) The proteins precipitated by a specific biotin-labeled circSamd4 probe were resolved by SDS-PAGE; bottom, the Vcp protein was detected by western blotting. CircSamd4 probe, biotin-labeled probe targeting circSamd4 back-splicing sequence; control probe, non-labeled probe targeting circSamd4 back-splicing sequence. (B) RIP assays revealed that the Vcp protein enriched circSamd4 in P1 CMs; ∗p < 0.05, n = 6. (C) qPCR assays were performed to detect the Vcp mRNA expression levels after circSamd4 knockdown; n = 6. (D) Western blot analysis of the Vcp protein levels in CMs. P1 CMs were transduced with Adv for 24 h and then incubated with H2O2 (20 μM) for 8 h, ∗p < 0.05, n = 6. (E) Western blot analysis of the Vcp protein levels in the cytoplasmic and mitochondrial fractions of CMs. ∗p < 0.05, n = 6. (F) Western blot analysis of the Vcp protein levels in the cytoplasmic and mitochondrial fractions of CMs. ∗p < 0.05, n = 6. (G) Western blot analysis of the Vcp protein levels in the cytoplasmic and mitochondrial fractions of CMs. ∗p < 0.05, n = 6. (H) Evaluation of P1 CM proliferative activity by Ki67 immunostaining after Adv-Vcp or Adv-NC transduction. Ki67+ CMs are indicated by arrows, ∗p < 0.05, n = 6. (I) Evaluation of P1 CM proliferative activity by pH3 immunostaining after Adv-Vcp or Adv-NC transduction. pH3+ CMs are indicated by arrows, ∗p < 0.05, n = 6. (J) Evaluation of the MMP in P1 CMs after Adv-Vcp or Adv-NC transduction. ∗p < 0.05, n = 6. JC-1 monomer, green; J-aggregate, red. (K) Evaluation of mitochondria-derived ROS levels in P1 CMs after Adv-Vcp or Adv-NC transduction. ∗p < 0.05, n = 6. (L) Evaluation of oxidative DNA damage in P1 CMs after Adv-Vcp or Adv-NC transduction. ∗p < 0.05, n = 6. (M) Detection of intracellular ROS levels in the myocardium 14 days after AAV9-circSamd4 transduction or ML240 treatment; ∗p < 0.05, n = 6. (N) Detection of oxidative DNA damage in adult CMs 14 days after AAV9-circSamd4 transduction or ML240 treatment; ∗p < 0.05, n = 6. (O) Evaluation of CM proliferation by pH3 immunostaining 14 days after AAV9-circSamd4 transduction or ML240 treatment; ∗p < 0.05, n = 6. (P) Masson trichrome staining of cross sections from adult mouse hearts 28 days after AAV9-circSamd4 transduction or ML240 treatment; ∗p < 0.05, n = 6.