phospho ser65 park2  (Bioss)

Journal: Communications Biology

Article Title: Pitavastatin activates mitophagy to protect EPC proliferation through a calcium-dependent CAMK1-PINK1 pathway in atherosclerotic mice

doi: 10.1038/s42003-022-03081-w

Figure Lengend Snippet: a Western blots revealed that MAP1LC3B-II turnover decreased ( b ) and the expression of SQSTM1 increased ( c ) significantly in atherosclerotic mice. e Representative western blots of mitophagic proteins in EPCs of atherosclerotic mice. ( d , f ) Quantitative analysis indicated that both the expression of PINK1 and PARK2 decreased on the mitochondrial membrane in EPCs of atherosclerotic mice. ( g , h ) Quantitative analysis revealed that MFN and BNIP3 showed no markedly difference in EPCs between ND and atherosclerotic mice. i Representative images and quantitative analysis of yellow and free red puncta in merged images in EPCs from ND and atherosclerotic mice, scale bar: 10 μm. j EPCs from ND and atherosclerotic mice were transfected by mtKeima for 12 h. EPCs from HFD8w and HFD16w ApoE −/− mice showed a significant lower mitophagy index in comparison to those from ND mice. Scale bar: 25 μm. (EPCs were isolated from ND, HFD8w and HFD16w ApoE −/− mice, n = 10 cells per group, cells were isolated from 3 mice for 1 experiment, and 3 independent experiments were performed, mean ± SD, * P < 0.05, ** P < 0.01).

Article Snippet: Antibody for phospho-Ser 65 -PARK2 (bs-19882R) was purchased from Bioss.

Techniques: Western Blot, Expressing, Transfection, Isolation

Journal: Communications Biology

Article Title: Pitavastatin activates mitophagy to protect EPC proliferation through a calcium-dependent CAMK1-PINK1 pathway in atherosclerotic mice

doi: 10.1038/s42003-022-03081-w

Figure Lengend Snippet: a Representative western blots for the detection of ATG7, ATG12–ATG5 conjugate, MAP1LC3B-II, and SQSTM1 after infection showed that Atg7 was successfully knocked down and mitophagy was effectively inhibited in Atg7 silencing group. b Silencing Atg7 before PTV treatment significantly reduced proliferative activity compared with PTV alone group. c 3-MA (2 mM) was added to inhibit mitophagy before 0.5 μM PTV treatment. CCK-8 assay showed that the proliferative activity in 3-MA + PTV group reduced significantly compared with PTV alone group. ( d – f ) PTV treatment (0, 0.1, 0.5, or 1.0 μM) for 24 h increased PINK1 accumulation and PARK2 recruitment in mitochondrial membrane in dose-dependent manner. g Mitotracker Deep Red was used to mark mitochondria and immunostain was employed to mark PARK2 in atherosclerotic mice EPCs. Merged images and Pearson’s overlap coefficient analysis indicated that PARK2 mostly localized on mitochondria in PTV treatment EPCs related to HFD group, scale bar: 10 μm. h Atherosclerotic mice EPCs were co-immunostained for MAP1LC3B and PARK2. Overlap of PARK2 with MAP1LC3B were observed in PTV treatment EPCs in comparison to HFD group and Pearson’s overlap coefficient was employed to analyze the co-localization, scale bar: 10 μm. (n = 10 cells per group, EPCs were isolated from ApoE −/− mice fed with high-fat diet for 8 weeks, cells were isolated from 3 mice for 1 experiment and 3 independent experiments were performed, mean ± SD, * P < 0.05, ** P < 0.01).

Article Snippet: Antibody for phospho-Ser 65 -PARK2 (bs-19882R) was purchased from Bioss.

Techniques: Western Blot, Infection, Activity Assay, CCK-8 Assay, Isolation

Journal: Communications Biology

Article Title: Pitavastatin activates mitophagy to protect EPC proliferation through a calcium-dependent CAMK1-PINK1 pathway in atherosclerotic mice

doi: 10.1038/s42003-022-03081-w

Figure Lengend Snippet: a Representative western blots showed that shRNA targeting Pink1 (sh Pink1 ) effectively silenced PINK1 protein expression and shRNA targeting Park2 (sh Park2 ) effectively silenced PARK2 protein expression after 24 h infection, respectively. Representative western blots and quantitative analysis revealed that either silencing Pink1 ( b , d ) or Park2 ( c , e ) significantly reduced MAP1LC3B expression. CCK-8 assay showed that either silencing Pink1 ( f ) or Park2 ( g ) before PTV treatment significantly reduced proliferative activity compared with PTV alone group. h EPCs of silencing Pink1 were seeded on E-plates for 24 h, 0.5 μM PTV was added to E-plates in PTV and sh Pink1 + PTV groups after 24 h incubation (black arrow). The normalized cell index indicated that EPC proliferative activity decreased in sh Pink1 + PTV group compared with PTV alone group. i EPCs of silencing Park2 were seeded on E-plates for 24 h, 0.5 μM PTV was added to E-plates in PTV and sh Park2 + PTV groups after 24 h incubation (black arrow). The normalized cell index indicated that EPC proliferative activity decreased in sh Park2 + PTV group compared with PTV alone group. j Western blots of the expression of PINK1, PARK2 and their phosphorylated form in WT and Pink1 −/− mice. EPCs with or without 0.5 μM PTV treatment were seeded on E-plates for 24 h, 0.5 μM in PTV and Pink1 −/− + PTV groups after 24 h incubation. The normalized cell index ( k ) and CCK-8 ( l ) indicated that EPC proliferative activity fell down in WT and Pink1 −/− mice with or without PTV treatment. (EPCs were isolated from ApoE −/− mice fed with high-fat diet for 8 weeks, cells were isolated from 3 mice for 1 experiment and 3 independent experiments were performed, mean ± SD, * P < 0.05, ** P < 0.01).

Article Snippet: Antibody for phospho-Ser 65 -PARK2 (bs-19882R) was purchased from Bioss.

Techniques: Western Blot, shRNA, Expressing, Infection, CCK-8 Assay, Activity Assay, Incubation, Isolation

Journal: Communications Biology

Article Title: Pitavastatin activates mitophagy to protect EPC proliferation through a calcium-dependent CAMK1-PINK1 pathway in atherosclerotic mice

doi: 10.1038/s42003-022-03081-w

Figure Lengend Snippet: a Immunoblot was employed to detect phosphorylation of PINK1 Ser 228 and PARK2 Ser 65 in each group. We normalized the expression of total PINK1 or PARK2 to compare the phosphorylation level. b Quantitative analysis revealed that PTV significantly up-regulated Ser 228 phosphorylation in PINK1 but Camk1 knockdown remarkably reduced this effect in PECs from atherosclerotic mice. c Quantitative analysis revealed that PTV significantly increased PARK2 Ser 65 phosphorylation but Camk1 knockdown reversed this effect in PECs from atherosclerotic mice. d PTV treatment reversed HFD8w-induced mitochondrial swelling and rupture of mitochondrial cristae; either PINK1 or ATG7 silencing blocked the effect of PTV on mitochondria of EPCs. e PTV treatment reversed HFD8w-induced ROS production, scale bar: 10 μm. f PTV treatment reversed HFD 8w-induced MMP decreased, scale bar: 10 μm. Quantitative analysis indicated the effect of PTV on ROS ( g ) and MMP ( h ) of EPCs can be inhibited by silencing ATG7, PINK1 blocked, and 3-MA pretreatment. (EPCs were isolated from 3 ApoE −/− mice fed with high fat diet for 8 weeks and 3 independent experiments were performed, mean ± SD, * P < 0.05, ** P < 0.01).

Article Snippet: Antibody for phospho-Ser 65 -PARK2 (bs-19882R) was purchased from Bioss.

Techniques: Western Blot, Expressing, Isolation

Journal: Communications Biology

Article Title: Pitavastatin activates mitophagy to protect EPC proliferation through a calcium-dependent CAMK1-PINK1 pathway in atherosclerotic mice

doi: 10.1038/s42003-022-03081-w

Figure Lengend Snippet: a EPCs tracing in vivo. pEGFP-N2-EPCs were pre-treated by 0.5 μM PTV for 24 h and labeled by acLDL-DiI. EPCs in PTV + shAtg7 group were received lentivirus transfection to knock down Atg7, and EPCs in PTV + shAtg7 groups were received lentivirus transfection to knock down Camk1, respectively. Then EPCs were injected into ApoE -/- mice after vascular injury and attached to the vascular injury site. EPCs homed to vascular endothelium exhibited both red (acLDL-DiI) and green (EGFP) fluorescence. More EPCs in PTV + VC group home to vascular injury site compared with those in PTV + shAtg7 and PTV + shCAMK1 groups. b Quantification of Evans blue staining showed that knocked down Atg7 (49.63 ± 6.20 %) or Camk1 (48.70 ± 6.62%) reduced reendothelialization area compared with those in PTV + VC groups (74.73 ± 5.68 %). VC, vector control. (EPCs were isolated from 3 normal mice. The figures represented 3 independent experiments; mean ± SD, ** P < 0.01). c A schematic drawing of the calcium signal and mitophagy pathway in EPCs after PTV treatment. PTV elicits mitochondrial calcium release and increases [Ca 2+ ] i that further phosphorylates CAMK1. CAMK1 phosphorylation serves as a PINK1 kinase to activate PINK1 and recruit as well as phosphorylates PARK2 to induce mitophagy. Mitophagy activation protects EPC proliferation by reducing mitochondrial ROS production and maintaining mitochondrial homeostasis in atherosclerosis.

Article Snippet: Antibody for phospho-Ser 65 -PARK2 (bs-19882R) was purchased from Bioss.

Techniques: In Vivo, Labeling, Transfection, Injection, Fluorescence, Staining, Plasmid Preparation, Isolation, Activation Assay