Journal: bioRxiv

Article Title: Deficiency of mitophagy mediator Parkin in aortic smooth muscle cells exacerbates abdominal aortic aneurysm

doi: 10.1101/2024.10.30.621201

Figure Lengend Snippet: (A) Pseudocolor plots for visualizing the mitophagy levels by gating the cells with lower MFI ratio of GFP versus mCherry. Cells are primary VSMCs isolated from Mito-QC mice, treated with 100nM, 500nM and 1000nM AngII (n=3 per group). (B) The MFI of MitoSox Red in MOVAS treated with 100nM, 500nM, and 1000nM AngII (n=6 per group). (C-E) The MFI of Mtphagy dye (n=7 per group) and MitoSox Red (n=6 per group) within MOVAS respectively treated with vehicle, 100nM AngII, or a combination of 100nM AngII with 100nM VB-08, were quantified and showed in a histogram, same for the MFI of Mitotracker and TMRM. The MFI ratio of TMRM to Mitotracker was employed as a metric for assessing the proportion of healthy mitochondria within the cells. (F-G) Alteration of protein levels in MOVAS after treatment with AngII alone or AngII in combination with VB-08, including PGC1α, Parkin, LC3I/II, were determined by Western blot (n=4 per group). β-tubulin is the internal control for normalization. (H) For MOVAS transfected with lentiviral scrambled or Parkin shRNA, the MFI of MitoSox Red within cells treated with vehicle, 100nM AngII, or a combination of 100nM AngII with 100nM VB-08 (n=12 per group), were quantified, same for the MFI ratio of TMRM to Mitotracker. Results are presented as mean ± SEM. Unpaired two-tailed Student’s t-test or Mann-Whitney U test were used for statistical analysis. Two-way ANOVA was used for H .

Article Snippet: Cells were then transduced with Parkin shRNA or control lentiviral particles at a multiplicity of infection (MOI) of 0.25, 0.5, or 1, in the presence of 5 µg/mL polybrene (Santa Cruz, catalog #sc-134220).

Techniques: Isolation, Western Blot, Control, Transfection, shRNA, Two Tailed Test, MANN-WHITNEY

Journal: Biomedicines

Article Title: Preservation of Mitochondrial Function by SkQ1 in Skin Fibroblasts Derived from Patients with Leber’s Hereditary Optic Neuropathy Is Associated with the PINK1/PRKN-Mediated Mitophagy

doi: 10.3390/biomedicines12092020

Figure Lengend Snippet: SkQ1 regulated PINK1/PRKN-mediated mitophagy. ( A – F ) LHON-IFBs were pretreated with SkQ1 (20 nM) and DMSO for 7 days, followed by treatment with 200 μM H 2 O 2 for 6 h. The expressions of PINK1, PRKN, LC3B, SOD2, VDAC1, and β-actin protein were detected by Western blot. β-actin was used as a reference, n ≥ 4. ( G , H ) The expression levels of PINK1 and LC3B in HEK293T and RPE-1 cells were consistent with those of LHON-IFBs, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, n ≥ 3. ( I ) SkQ1 could be involved in regulating oxidative stress and PINK1/PRKN-mediated mitophagy pathway.

Article Snippet: After blocking with 5% milk powder (PS112L, Epizyme Biomedical Technology) for 1.5 h, the membranes were incubated with primary antibodies overnight at 4 °C: PINK1 (6946S, CST, Massachusetts, MA, USA), PRKN (32833, CST, MA, USA), SOD2 (ET1701-54, HuaBio, Hangzhou, China), VDAC1 (ET1601-20, HuaBio, Hangzhou, China), LC3B (ET1701-65, Hangzhou, China) and β-actin (3700S, CST, MA, USA).

Techniques: Western Blot, Expressing