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

Journal: Nature Communications

Article Title: Omegasome-proximal PtdIns(4,5)P 2 couples F-actin mediated mitoaggregate disassembly with autophagosome formation during mitophagy

doi: 10.1038/s41467-019-08924-5

Figure Lengend Snippet: Synchronous formation between BATS and circular F-actin structures during mitoaggregate disassembly. a A MitoTracker DeepRed FM-stained HeLa cell co-expressing EBFP2-Parkin and hrGFP-BATS was illuminated with 635 nm (white circle) to locally activate Parkin-mediated mitophagy. b Selected frames from a time-lapse movie (Supplementary Movie ) on the cellular region undergoing mitophagy in a after 635 nm illumination. Scale bar: 10 μm. c Two examples of synchronous formation of BATS and circular F-actin structures near Parkin-labeled mitoaggregates. MitoTracker DeepRed FM-stained HeLa cells co-expressing EBFP2-Parkin, LifeAct-EGFP, and TagRFP-BATS were illuminated with 635 nm to locally activate Parkin-mediated mitophagy. Scale bar: 1 μm. d Newly formed circular F-actin (green lines) and BATS-positive structures (red lines) are indicated on thresholded images from a time-lapse movie (Supplementary Movie ). Circular F-actin and BATS structures formation overlapped greater than 80% in time as shown in the graph ( n = 20 from five independent experiments). e Mitoaggregate-associated BATS sites are decreased under CK666 treatment. Total BATS counts near Parkin-labeled mitochondria were calculated from 200 min time-lapse images with 2 min interval (mean ± S.D.; control n = 11, CK666 n = 11 biologically independent samples; *** p < 0.0001 as evaluated using two-tailed unpaired Student’s t -test)

Article Snippet: EBFP2-Parkin and TagRFP-Parkin were constructed by subcloning EBFP2 (Addgene, 14893) and TagRFP (Evrogen, FP141) into EYFP-Parkin (Addgene, 23955) respectively.

Techniques: Staining, Expressing, Labeling, Control, Two Tailed Test

Journal: Nature Communications

Article Title: Omegasome-proximal PtdIns(4,5)P 2 couples F-actin mediated mitoaggregate disassembly with autophagosome formation during mitophagy

doi: 10.1038/s41467-019-08924-5

Figure Lengend Snippet: Non-disassembled mitoaggregates harbored reduced autophagosome generation and displayed delayed turnover. a Diagram illustrating the principle underlying photoclustering experiments. After 635 nm illumination, CRY2-Ub become conjugated to damaged mitochondria through Parkin catalyzed ubiquitination. 488 nm illumination drives CRY2 oligomerization, which helps to maintain the clustering of damaged mitochondria and delays disassembly. For clarity, representative CRY2-Ub were drawn. b A MitoTracker DeepRed FM-stained HeLa cell co-expressing EBFP2-Parkin, CRY2-EGFP-Ub, and TPC2-TagRFP was illuminated with 635 nm to locally activate Parkin-mediated mitophagy. Selected frames of mitoaggregates of interest 40–340 min after mitophagy initiation are shown. During mitophagy, a portion of the mitoaggregates were 488-nm illuminated (as indicated by white dotted circles) at both 80 and 140 min. c The progression of mitophagy in b 340 min after mitochondrial damage. EGFP-quenched and TPC2-positive Parkin-labeled mitochondria represents mature autophagolysosomes (yellow arrows). EGFP-fluorescence retaining and TPC2-negative Parkin-labeled mitochondria represents ubiquitinated mitochondria outside of lysosomes (blue arrows). Photoclustered mitoaggregates are outlined with a blue-dotted line. d Diagram illustrating the experimental flow of photoclustering experiments in single cells. e A MitoTracker DeepRed FM-stained HeLa cell co-expressing EBFP2-Parkin, CRY2-TagRFP-Ub, and hrGFP-BATS was illuminated with 635 nm to activate Parkin-mediated mitophagy at two separate locations (white circles) within a single cell. f Selected frames from a time-lapse movie (Supplementary Movie ) on cellular region containing mitoaggregates in e 0–366 min after 635 nm illumination. 488 nm mediated clustering (green dotted circles) were triggered at both 77 and 154 min. The red arrows denote BATS-positive structures on the surfaces of photoclustered mitoaggregates. g Total number of BATS puncta in non 488-nm illuminated and 488-nm illuminated regions as shown in f . Scale bar: 10 μm

Article Snippet: EBFP2-Parkin and TagRFP-Parkin were constructed by subcloning EBFP2 (Addgene, 14893) and TagRFP (Evrogen, FP141) into EYFP-Parkin (Addgene, 23955) respectively.

Techniques: Ubiquitin Proteomics, Staining, Expressing, Labeling, Fluorescence

Journal: Italian Journal of Animal Science

Article Title: Pterostilbene attenuates oxidative stress induced by hydrogen peroxide in MAC-T cells through activating PINK1/Parkin-mediated mitophagy

doi: 10.1080/1828051x.2024.2353853

Figure Lengend Snippet: Figure 3. Pterostilbene (PTE) attenuated H2O2-triggered oxidative damage to MAC-T cells. Pre-treatment with PTE (10 lM) was performed for 12 h, followed by H2O2 (600 lM) treatment of MAC-T cells for 24 h. (a) Representative Western blot bands for HO-1, SOD1 and GPX4 proteins. (b) Quantitation of HO-1 protein level in MAC-T cells. (c) Quantitation of SOD1 protein level in MAC-T cells. (d) Quantitation of GPX4 protein level in MAC-T. (e) Quantitation of NRF2 protein level in MAC-T data are present as mean ± SEM (n ¼ 3), p < 0.01 versus control group, ##p < 0.01 versus H2O2 group.

Article Snippet: Subsequently, the membranes were blocked with 3% defatted milk powder (P0216, Beyotime, China) for 2 h at room temperature and incubated with primary antibodies at 4 �C overnight against HO-1 (27282-1-AP, Proteintech, China), SOD1 (10269-1-AP, Proteintech), GPX4 (14432- 1-AP, Proteintech), Parkin (14060-1-AP, Proteintech), PINK1 (23274-1-AP, Proteintech), beclin-1 (11306-1-AP, Proteintech) and GAPDH (60004-1-Ig, Proteintech).

Techniques: Western Blot, Quantitation Assay, Control