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

Journal: Autophagy

Article Title: The TOMM machinery is a molecular switch in PINK1 and PARK2/PARKIN-dependent mitochondrial clearance.

doi: 10.4161/auto.25884

Figure Lengend Snippet: Figure 1. Impaired mitochondrial import triggers PARK2 recruitment to mitochondria. (A) Left panels: mitochondrial redistribution of PARK2 in models of defective mitochondrial protein import. Mitochondria were visualized with MitoTracker CMXRos and anti-VDAC1 staining. Treatments are indicated in italics. Right panels: mitochondrial import impairment of mitoGFP (western blots) and quantification of the abundance of the imported mitoGFP protein in total lysates from CCCP-treated cells, or in mitochondrion-enriched fractions subjected to trypsin digestion in cells with lower TOMM40 levels (n = 3 independent experiments). Loading controls: ATP synthase subunit α (ATP5A1), ACTA1/actin (actin, α 1, skeletal muscle). Note the disappearance of the TOMM70A immunoreactive band in trypsin-digested mitochondrial fractions. (B) Proportion of cells with colocalization of PARK2 and VDAC1 immunostainings in the various models. (C) Fluorescence intensity of TMRM in live cells. n = 3 independent wells from one of at least three independent experiments. *P < 0.05, ***P < 0.001 vs. the corresponding control condition. Scale bar: 10 µm.

Article Snippet: The Table 2. antibodies used for immunocytochemical staining Primary antibodies Type Company Catalog # Host Dilution PaRK2, clone Park8 Millipore MaB5512 mouse 1:1000 TOMM20 abcam ab56783 mouse 1:1000 TOMM20 santa cruz sc-11415 rabbit 1:500 TOMM22 abcam ab10436 mouse 1:5000 TOMM22 atlas antibodies hPa003037 rabbit 1:1000 TOMM22 santa cruz sc-14894 goat 1:200 cYcs, clone 7h8.2c12 BD PharMingen 556432 mouse 1:1000 hsD17B10 abcam ab10260 mouse 1:2000 PaRK2 Millipore MaB5112 rabbit 1:7000 TOMM70a sigma hPa014589 rabbit 1:200 TOMM70a abcam ab106193 mouse 1:2000 TOMM40 santa cruz sc-11414 rabbit 1:100 TOMM40 clone D2 santa cruz sc-365467 mouse 1:200 TOMM40 santa cruz sc-11022 goat 1:100 PMPcB Proteintech 16064-1-aP rabbit 1:400 VDac1 abcam ab15895 rabbit 1:200 LONP1 (G. isaya, Mayo clinic Foundation Rochester, MN) - - rabbit 1:3000 PiNK1 Novus Biologicals Bc100-494 rabbit 1:2500 aTG5 Novus Biologicals NB110-53818 rabbit 1:500 aTG7 abcam ab53255 rabbit 1:500 aMBRa1 Novus Biologicals FLJ20294 rabbit 1:250 Secondary antibodies alexa Fluor 488 anti-mouse/rabbit invitrogen a11070/a11029 goat 1:2000; 1:10000* alexa Fluor 568 anti-mouse/rabbit invitrogen a11036/a11031 goat 1:10000*; 1:30000* alexa Fluor 633 anti-mouse/rabbit invitrogen a21050/a21070 goat 1:500 cY3-conjugated anti-mouse/rabbit sigma c2181/c2306 sheep 1:2000 *Dilution used for FReT and FLiM studies D ow nl oa de d by [ A rc hi ve s & B ib lio th èq ue s de l' U L B ] at 1 2: 09 0 6 Fe br ua ry 2 01 5 1816 autophagy Volume 9 issue 11 Mann-Whitney U test was used to compare TMRM intensities between conditions within each cell type (Fig. 1C).

Techniques: Staining, Western Blot, Fluorescence, Control

Journal: Autophagy

Article Title: The TOMM machinery is a molecular switch in PINK1 and PARK2/PARKIN-dependent mitochondrial clearance.

doi: 10.4161/auto.25884

Figure Lengend Snippet: Figure 2. PARK2 interacts with the TOMM machinery in a PINK1-dependent manner. (A) Representative FRET images for the indicated donor (green)- acceptor (red) pairs in HEK293T cells with low TOMM40 levels. Pseudocolor scale: FRET efficiencies reflecting individual pixel donor fluorescence changes after acceptor photobleaching. E: mean FRET efficiency within the region of interest (ROI, yellow frame). N: nucleus. (B) Quantitative FRET analysis and percentage of cells with FRET for all donor-acceptor pairs analyzed in HEK293T cells transfected with siRNA against TOMM40 or treated with CCCP. n = 10 to 35 cells from two independent experiments. Comparisons were made with VDAC1-PARK2 for donor-PARK2 pairs, PINK1-PARK2 in the corresponding “Control siRNA” or “CCCP-” conditions for PINK1-acceptor pairs. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar: 3 µm.

Article Snippet: The Table 2. antibodies used for immunocytochemical staining Primary antibodies Type Company Catalog # Host Dilution PaRK2, clone Park8 Millipore MaB5512 mouse 1:1000 TOMM20 abcam ab56783 mouse 1:1000 TOMM20 santa cruz sc-11415 rabbit 1:500 TOMM22 abcam ab10436 mouse 1:5000 TOMM22 atlas antibodies hPa003037 rabbit 1:1000 TOMM22 santa cruz sc-14894 goat 1:200 cYcs, clone 7h8.2c12 BD PharMingen 556432 mouse 1:1000 hsD17B10 abcam ab10260 mouse 1:2000 PaRK2 Millipore MaB5112 rabbit 1:7000 TOMM70a sigma hPa014589 rabbit 1:200 TOMM70a abcam ab106193 mouse 1:2000 TOMM40 santa cruz sc-11414 rabbit 1:100 TOMM40 clone D2 santa cruz sc-365467 mouse 1:200 TOMM40 santa cruz sc-11022 goat 1:100 PMPcB Proteintech 16064-1-aP rabbit 1:400 VDac1 abcam ab15895 rabbit 1:200 LONP1 (G. isaya, Mayo clinic Foundation Rochester, MN) - - rabbit 1:3000 PiNK1 Novus Biologicals Bc100-494 rabbit 1:2500 aTG5 Novus Biologicals NB110-53818 rabbit 1:500 aTG7 abcam ab53255 rabbit 1:500 aMBRa1 Novus Biologicals FLJ20294 rabbit 1:250 Secondary antibodies alexa Fluor 488 anti-mouse/rabbit invitrogen a11070/a11029 goat 1:2000; 1:10000* alexa Fluor 568 anti-mouse/rabbit invitrogen a11036/a11031 goat 1:10000*; 1:30000* alexa Fluor 633 anti-mouse/rabbit invitrogen a21050/a21070 goat 1:500 cY3-conjugated anti-mouse/rabbit sigma c2181/c2306 sheep 1:2000 *Dilution used for FReT and FLiM studies D ow nl oa de d by [ A rc hi ve s & B ib lio th èq ue s de l' U L B ] at 1 2: 09 0 6 Fe br ua ry 2 01 5 1816 autophagy Volume 9 issue 11 Mann-Whitney U test was used to compare TMRM intensities between conditions within each cell type (Fig. 1C).

Techniques: Fluorescence, Transfection, Control

Journal: Autophagy

Article Title: The TOMM machinery is a molecular switch in PINK1 and PARK2/PARKIN-dependent mitochondrial clearance.

doi: 10.4161/auto.25884

Figure Lengend Snippet: Figure 5. Depletion of TOMM core complex subunits bypasses the requirement for PINK1, but not PARK2, in mitochondrial clearance. (A) Immunostaining and (B) corresponding quantifications illustrating the effect of PINK1 depletion on the fate of VDAC1 and PMPCB in COS7 cells with low TOMM40 levels, with or without exogenous PARK2, or in cells with exogenous PARK2 only. Arrows: loss of mitochondrial markers. n = 3 independent wells from one experiment representative of five. (C) Images illustrating the relative loss of PMPCB immunostaining in representative conditions. The right panels show PMPCB staining after application of a threshold mask, with A corresponding to mitochondrial area relative to total cell area (%). (D and E, left graph) Quantification of the cell area covered by PMPCB (relative mitochondrial area), estimated as in (C), in the indicated conditions. n = 3 independent wells from one experiment representative of three. (E, right graph) Effect of siRNA-mediated silencing of endogenous PARK2, estimated by real-time PCR with two different primer pairs on mRNA extracted from COS7 cells and retrotranscribed into cDNA. n = 3 independent wells from one representative experi- ment out of two. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the corresponding control condition: “+ PARK2” in (B),” Control siRNA” (D and E). aP < 0.001 vs. the corresponding “TOMM40 siRNA” condition in (B). Scale bar: 10 µm.

Article Snippet: The Table 2. antibodies used for immunocytochemical staining Primary antibodies Type Company Catalog # Host Dilution PaRK2, clone Park8 Millipore MaB5512 mouse 1:1000 TOMM20 abcam ab56783 mouse 1:1000 TOMM20 santa cruz sc-11415 rabbit 1:500 TOMM22 abcam ab10436 mouse 1:5000 TOMM22 atlas antibodies hPa003037 rabbit 1:1000 TOMM22 santa cruz sc-14894 goat 1:200 cYcs, clone 7h8.2c12 BD PharMingen 556432 mouse 1:1000 hsD17B10 abcam ab10260 mouse 1:2000 PaRK2 Millipore MaB5112 rabbit 1:7000 TOMM70a sigma hPa014589 rabbit 1:200 TOMM70a abcam ab106193 mouse 1:2000 TOMM40 santa cruz sc-11414 rabbit 1:100 TOMM40 clone D2 santa cruz sc-365467 mouse 1:200 TOMM40 santa cruz sc-11022 goat 1:100 PMPcB Proteintech 16064-1-aP rabbit 1:400 VDac1 abcam ab15895 rabbit 1:200 LONP1 (G. isaya, Mayo clinic Foundation Rochester, MN) - - rabbit 1:3000 PiNK1 Novus Biologicals Bc100-494 rabbit 1:2500 aTG5 Novus Biologicals NB110-53818 rabbit 1:500 aTG7 abcam ab53255 rabbit 1:500 aMBRa1 Novus Biologicals FLJ20294 rabbit 1:250 Secondary antibodies alexa Fluor 488 anti-mouse/rabbit invitrogen a11070/a11029 goat 1:2000; 1:10000* alexa Fluor 568 anti-mouse/rabbit invitrogen a11036/a11031 goat 1:10000*; 1:30000* alexa Fluor 633 anti-mouse/rabbit invitrogen a21050/a21070 goat 1:500 cY3-conjugated anti-mouse/rabbit sigma c2181/c2306 sheep 1:2000 *Dilution used for FReT and FLiM studies D ow nl oa de d by [ A rc hi ve s & B ib lio th èq ue s de l' U L B ] at 1 2: 09 0 6 Fe br ua ry 2 01 5 1816 autophagy Volume 9 issue 11 Mann-Whitney U test was used to compare TMRM intensities between conditions within each cell type (Fig. 1C).

Techniques: Immunostaining, Staining, Real-time Polymerase Chain Reaction, Control

Journal: Autophagy

Article Title: The TOMM machinery is a molecular switch in PINK1 and PARK2/PARKIN-dependent mitochondrial clearance.

doi: 10.4161/auto.25884

Figure Lengend Snippet: Figure 6. PMPCB downregulation does not prime mitochondria for OMM clearance. Effect of PMPCB downregulation on the clearance of VDAC1 and HSD17B10 (matrix marker) in COS7 cells with or without PINK1 or exogenous PARK2. n = 3 independent wells from one experiment representative of three. ***P < 0.001 vs. the corresponding “+ PARK2 condition”. Scale bar: 10 µm.

Article Snippet: The Table 2. antibodies used for immunocytochemical staining Primary antibodies Type Company Catalog # Host Dilution PaRK2, clone Park8 Millipore MaB5512 mouse 1:1000 TOMM20 abcam ab56783 mouse 1:1000 TOMM20 santa cruz sc-11415 rabbit 1:500 TOMM22 abcam ab10436 mouse 1:5000 TOMM22 atlas antibodies hPa003037 rabbit 1:1000 TOMM22 santa cruz sc-14894 goat 1:200 cYcs, clone 7h8.2c12 BD PharMingen 556432 mouse 1:1000 hsD17B10 abcam ab10260 mouse 1:2000 PaRK2 Millipore MaB5112 rabbit 1:7000 TOMM70a sigma hPa014589 rabbit 1:200 TOMM70a abcam ab106193 mouse 1:2000 TOMM40 santa cruz sc-11414 rabbit 1:100 TOMM40 clone D2 santa cruz sc-365467 mouse 1:200 TOMM40 santa cruz sc-11022 goat 1:100 PMPcB Proteintech 16064-1-aP rabbit 1:400 VDac1 abcam ab15895 rabbit 1:200 LONP1 (G. isaya, Mayo clinic Foundation Rochester, MN) - - rabbit 1:3000 PiNK1 Novus Biologicals Bc100-494 rabbit 1:2500 aTG5 Novus Biologicals NB110-53818 rabbit 1:500 aTG7 abcam ab53255 rabbit 1:500 aMBRa1 Novus Biologicals FLJ20294 rabbit 1:250 Secondary antibodies alexa Fluor 488 anti-mouse/rabbit invitrogen a11070/a11029 goat 1:2000; 1:10000* alexa Fluor 568 anti-mouse/rabbit invitrogen a11036/a11031 goat 1:10000*; 1:30000* alexa Fluor 633 anti-mouse/rabbit invitrogen a21050/a21070 goat 1:500 cY3-conjugated anti-mouse/rabbit sigma c2181/c2306 sheep 1:2000 *Dilution used for FReT and FLiM studies D ow nl oa de d by [ A rc hi ve s & B ib lio th èq ue s de l' U L B ] at 1 2: 09 0 6 Fe br ua ry 2 01 5 1816 autophagy Volume 9 issue 11 Mann-Whitney U test was used to compare TMRM intensities between conditions within each cell type (Fig. 1C).

Techniques: Marker

Journal: Autophagy

Article Title: The TOMM machinery is a molecular switch in PINK1 and PARK2/PARKIN-dependent mitochondrial clearance.

doi: 10.4161/auto.25884

Figure Lengend Snippet: Figure 7. TOMM40 downregulation triggers PARK2-dependent mitophagy. (A) Representative images illustrating LC3-positive vesicles associated with PMPCB staining in COS7 cells, in the indicated conditions. Right graphs: quantification of the number of LC3-positive vesicles costained for PMPCB rela- tive to the total number of LC3 vesicles in cells, as explained in the Materials and Methods section. The control conditions were arbitrarily set at 1. n = 20 cells from three independent experiments. (B) Quantification of the proportion of cells without VDAC1 or PMPCB staining after overproduction of PARK2 or downregulation of TOMM40, and silencing of ATG5, ATG7, or AMBRA1, as indicated. Cells were treated with CCCP in all conditions. n = 3 independent wells from one experiment representative of three. (C) Images showing the relative loss of PMPCB immunostaining and (graph) quantification of the rela- tive mitochondrial area covered by PMPCB staining, estimated in the indicated conditions as explained in the legend of Figure 5C. A: relative mitochon- drial area (%). n = 30 cells from one experiment representative of three. **P < 0.01, ***P < 0.001 vs. (A) the corresponding control condition within “CCCP +” or “CCCP -”; (B and C) the corresponding “Control siRNA” condition. aP < 0.01, bP < 0.001 vs. the corresponding “CCCP -” condition. Scale bar: 10 µm.

Article Snippet: The Table 2. antibodies used for immunocytochemical staining Primary antibodies Type Company Catalog # Host Dilution PaRK2, clone Park8 Millipore MaB5512 mouse 1:1000 TOMM20 abcam ab56783 mouse 1:1000 TOMM20 santa cruz sc-11415 rabbit 1:500 TOMM22 abcam ab10436 mouse 1:5000 TOMM22 atlas antibodies hPa003037 rabbit 1:1000 TOMM22 santa cruz sc-14894 goat 1:200 cYcs, clone 7h8.2c12 BD PharMingen 556432 mouse 1:1000 hsD17B10 abcam ab10260 mouse 1:2000 PaRK2 Millipore MaB5112 rabbit 1:7000 TOMM70a sigma hPa014589 rabbit 1:200 TOMM70a abcam ab106193 mouse 1:2000 TOMM40 santa cruz sc-11414 rabbit 1:100 TOMM40 clone D2 santa cruz sc-365467 mouse 1:200 TOMM40 santa cruz sc-11022 goat 1:100 PMPcB Proteintech 16064-1-aP rabbit 1:400 VDac1 abcam ab15895 rabbit 1:200 LONP1 (G. isaya, Mayo clinic Foundation Rochester, MN) - - rabbit 1:3000 PiNK1 Novus Biologicals Bc100-494 rabbit 1:2500 aTG5 Novus Biologicals NB110-53818 rabbit 1:500 aTG7 abcam ab53255 rabbit 1:500 aMBRa1 Novus Biologicals FLJ20294 rabbit 1:250 Secondary antibodies alexa Fluor 488 anti-mouse/rabbit invitrogen a11070/a11029 goat 1:2000; 1:10000* alexa Fluor 568 anti-mouse/rabbit invitrogen a11036/a11031 goat 1:10000*; 1:30000* alexa Fluor 633 anti-mouse/rabbit invitrogen a21050/a21070 goat 1:500 cY3-conjugated anti-mouse/rabbit sigma c2181/c2306 sheep 1:2000 *Dilution used for FReT and FLiM studies D ow nl oa de d by [ A rc hi ve s & B ib lio th èq ue s de l' U L B ] at 1 2: 09 0 6 Fe br ua ry 2 01 5 1816 autophagy Volume 9 issue 11 Mann-Whitney U test was used to compare TMRM intensities between conditions within each cell type (Fig. 1C).

Techniques: Staining, Control, Immunostaining

Journal: Autophagy

Article Title: The TOMM machinery is a molecular switch in PINK1 and PARK2/PARKIN-dependent mitochondrial clearance.

doi: 10.4161/auto.25884

Figure Lengend Snippet: Figure 8. Model illustrating putative steps in the mitochondrial clearance program mediated by the PINK1-PARK2 pathway. PINK1 is imported into healthy mitochondria through the TOMM and TIMM complexes, and processed by PMPC and the presenilin-associated rhomboid-like protease PARL (1).33 Loss of mitochondrial import efficiency, associated or not with ΔΨ collapse, leads to accumulation of PINK1 at the OMM, recruitment of PARK2 in proximity of the TOMM complex (2), destabilization of the machinery, possibly caused by proteasomal degradation of key subunits, OMM rupture and proteolysis (3) and elimination of whole mitochondria by the proteasome and lysosome-dependent pathways (4). PINK1 and PARK2 are both required to trigger step 3, whereas only PARK2 is essential for step 4.

Article Snippet: The Table 2. antibodies used for immunocytochemical staining Primary antibodies Type Company Catalog # Host Dilution PaRK2, clone Park8 Millipore MaB5512 mouse 1:1000 TOMM20 abcam ab56783 mouse 1:1000 TOMM20 santa cruz sc-11415 rabbit 1:500 TOMM22 abcam ab10436 mouse 1:5000 TOMM22 atlas antibodies hPa003037 rabbit 1:1000 TOMM22 santa cruz sc-14894 goat 1:200 cYcs, clone 7h8.2c12 BD PharMingen 556432 mouse 1:1000 hsD17B10 abcam ab10260 mouse 1:2000 PaRK2 Millipore MaB5112 rabbit 1:7000 TOMM70a sigma hPa014589 rabbit 1:200 TOMM70a abcam ab106193 mouse 1:2000 TOMM40 santa cruz sc-11414 rabbit 1:100 TOMM40 clone D2 santa cruz sc-365467 mouse 1:200 TOMM40 santa cruz sc-11022 goat 1:100 PMPcB Proteintech 16064-1-aP rabbit 1:400 VDac1 abcam ab15895 rabbit 1:200 LONP1 (G. isaya, Mayo clinic Foundation Rochester, MN) - - rabbit 1:3000 PiNK1 Novus Biologicals Bc100-494 rabbit 1:2500 aTG5 Novus Biologicals NB110-53818 rabbit 1:500 aTG7 abcam ab53255 rabbit 1:500 aMBRa1 Novus Biologicals FLJ20294 rabbit 1:250 Secondary antibodies alexa Fluor 488 anti-mouse/rabbit invitrogen a11070/a11029 goat 1:2000; 1:10000* alexa Fluor 568 anti-mouse/rabbit invitrogen a11036/a11031 goat 1:10000*; 1:30000* alexa Fluor 633 anti-mouse/rabbit invitrogen a21050/a21070 goat 1:500 cY3-conjugated anti-mouse/rabbit sigma c2181/c2306 sheep 1:2000 *Dilution used for FReT and FLiM studies D ow nl oa de d by [ A rc hi ve s & B ib lio th èq ue s de l' U L B ] at 1 2: 09 0 6 Fe br ua ry 2 01 5 1816 autophagy Volume 9 issue 11 Mann-Whitney U test was used to compare TMRM intensities between conditions within each cell type (Fig. 1C).

Techniques:

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Pink1/PARK2/mROS-Dependent Mitophagy Initiates the Sensitization of Cancer Cells to Radiation

doi: 10.1155/2021/5595652

Figure Lengend Snippet: Sarm1-mtKR-induced mtROS participated in the autophagic cell death depending on the Pink1/PARK2 pathway. (a) WB was performed to determine protein levels of Pink1, PARK2, and Tom20 in total- and mito-proteins. GAPDH and HSP60 proteins were used for loading control. (b) The gray ratios of Pink1, PARK2, and Tom20 in total- and mito-proteins. (c) Pink1 and Tom20 mRNAs detected by qRT-PCR. Bars represent mean ± SD of triplicate measurements. ∗ P < 0.05 and ∗∗ P < 0.01, versus control; # P < 0.05, versus light exposure.

Article Snippet: Anti-COX IV, anti- β -actin, and anti-GAPDH were purchased from Santa Cruz, CA, USA; anti-voltage-dependent anion channel 1 (VDAC1), anti-heat-shock protein 60 (HSP60), anti-Pink1, and anti-PARK2 were purchased from Bioworld Technology, Inc., USA; and anti-microtubule-associated protein 1 light chain 3 (LC3), anti-p62, and anti-Tom20 were purchased from Cell Signaling Technology, Danvers, MA, USA.

Techniques: Quantitative RT-PCR

Journal: Autophagy

Article Title: RAB7 activity is required for the regulation of mitophagy in oocyte meiosis and oocyte quality control during ovarian aging

doi: 10.1080/15548627.2021.1946739

Figure Lengend Snippet: Evaluation of mitophagy activity in oocytes during maturation. P23 female mice were injected with PMSG for 48 h and fully grown oocytes (GV stage) were collected for IVM. Oocytes were harvested at different meiotic stages: 0 h (GV), 3 h (GVBD), 8 h (Pro-MI or MI) and 12 h (MII). (A) Mitophagic flux reflected by mt-Keima labeling. GV oocytes were microinjected with mt-Keima mRNAs and hold in 2 μM milrinone for 8–10 h. Images of oocytes at different meiotic stages were collected at fluorescent emission 550 nm (red) and 440 nm (green), respectively. Red means the active mitophagic flux as the fusion of mitophagosome with lysosome. (B) Immunofluorescence of PRKN in oocytes at each meiotic stage. TUBB was used to label meiotic spindle formation and DNA was stained with Hoechst 33342. Red, PRKN; Green, TUBB; Pink, DNA. (C) Immunofluorescence of RAB7 in oocytes at each meiotic stage. Red, RAB7. Bars: 20 μm. (D) Western blots of RAB7, PRKN and PINK1 proteins in oocytes during meiotic progression. The expression of TUBB was used as internal control. (E) Relative intensity of RAB7, PINK1 and PRKN proteins as compared with TUBB. *, P < 0.05.

Article Snippet: Primary and secondary antibodies were obtained from the following commercial sources: anti-RAB7A antibody (Cell Signaling Technology, 95,746), anti-PRKN antibody (ZEN BIO, 381,626), anti-ATG9A antibody (ZEN BIO, 381,260), anti-LAMP1 antibody (DSHB, 1D4B), anti-POU5F1/OCT4 antibody (Abcam, ab25630) and anti-TOMM20 antibody (Santa Cruz Biotechnology, sc-17764), anti-TUBB (Cell Signaling Technology, 2146).

Techniques: Activity Assay, Injection, Labeling, Immunofluorescence, Staining, Western Blot, Expressing

Journal: Autophagy

Article Title: RAB7 activity is required for the regulation of mitophagy in oocyte meiosis and oocyte quality control during ovarian aging

doi: 10.1080/15548627.2021.1946739

Figure Lengend Snippet: Block of meiosis in CCCP treated oocytes. GV oocytes were collected and treated with or without 1 µM CCCP to follow the meiotic progression. (A) Decreased GVBD and first polar body extrusion (PB) rates after CCCP treatment. The GVBD rate was monitored in the first 3 hours and the PB rate was calculated from 8 h-12 h after CCCP treatment. n = 60 oocytes in each group. (B) Activation of mitophagy after CCCP treatment. 10 pl of 500 ng/ml mCherry-Prkn mRNAs were microinjected into GV oocytes and oocytes were hold in 2 μM milrinone for 2 h before treated with CCCP. Oocytes were collected at 0 h, 0.5 h, 1 h and 3 h for MitoTracker staining. Hoechst 33342 was used to label DNA. Red, PRKN; Green, mitochondria; Blue, Hoechst 33342. Bar: 20 µm. (C) CCCP induced mitochondria damage in oocytes and 293 T cell by TEM. Oocytes were collected at 0 h, 0.5 h and 1 h and 293 T cells were harvested at 0.5 h, 1 h and 3 h after treatment. Some oocytes treated with CCCP and RAB7 activator ML098 (1 µM) for 0.5 h were also collected for TEM analysis. Insets, representative good or damaged mitochondria. White arrows, mitochondria with good morphology; black arrows, vacuolated mitochondria. Bars: 500 nm. (D) Accumulation of mitochondria damage in oocytes or 293 T cells. Under TEM images, percentages of damaged mitochondria per area (500 nmX500 nm) were shown. At least three visions were chosen and mitochondria were counted by two individuals. (E) Western blots of RAB5, RAB7, PRKN and PINK1 in oocytes after CCCP treatment. (F) Relative intensity of proteins detected with western blots. TUBB was used as internal control. *, P < 0.05; **, P < 0.01 as compared with controls.

Article Snippet: Primary and secondary antibodies were obtained from the following commercial sources: anti-RAB7A antibody (Cell Signaling Technology, 95,746), anti-PRKN antibody (ZEN BIO, 381,626), anti-ATG9A antibody (ZEN BIO, 381,260), anti-LAMP1 antibody (DSHB, 1D4B), anti-POU5F1/OCT4 antibody (Abcam, ab25630) and anti-TOMM20 antibody (Santa Cruz Biotechnology, sc-17764), anti-TUBB (Cell Signaling Technology, 2146).

Techniques: Blocking Assay, Activation Assay, Staining, Western Blot

Journal: Autophagy

Article Title: RAB7 activity is required for the regulation of mitophagy in oocyte meiosis and oocyte quality control during ovarian aging

doi: 10.1080/15548627.2021.1946739

Figure Lengend Snippet: RAB7 knockdown leads to oocyte maturation disorder and abnormal mitophagy. GV oocytes were microinjected with 10 pl Rab7 siRNA (siRab7-1 and siRab7-2) or control siRNA (siCtrl) (500 ng/µl) and arrested at the GV stage for 24 h in M16 medium containing 2 μM milrinone. (A) Decreased GVBD and PB rates after Rab7 siRNA treatment. The GVBD rate was monitored in the first 3 hours and the PB rate was counted from 8 h-12 h after releasing from milrinone. n = 150 oocytes in each group. (B) Abnormal spindle morphology in oocytes after siRab7 treatment. Oocytes in control and treated group were collected at MI stage for immunofluorescence with TUBB antibody. Green, TUBB; Blue, DNA labeled with Hoechst 33342. The length and width of each spindle and the width of chromosome were measured (µm) and then they were shown as the ratio of length to width (length:width) and ratio of chromosome disperse to length (chromosome width:length). n = 20 oocytes in each group. Bar: 10 μm. (C) Chromosome spread in siCtrl and siRab7 treated oocytes. Chromosome was labeled with Hoechst 33342 (blue) and centrosome was labeled with SS18L1/CREST staining (red). Chromosome was counted with two individuals and aneuploidy rates were evaluated. n = 20 oocytes in each group. Bar: 10 μm. (D) Co-staining of ATG9 and LAMP1 in oocytes of siCtrl and siRab7 treated groups by immunofluorescence. Red, ATG9; Green, LAMP1; Pink, DNA labeled with Hoechst 33342. The association of ATG9-positive vesicles with LAMP1 labeled lysosomes was evaluated in control and treated oocytes. n = 20 oocytes in each group. Bar: 20 μm. (E) Immunofluorescence of PRKN in siCtrl and siRab7 treated oocytes. Double staining with TOMM20, the mitochondria outer membrane protein was performed to reveal the translocation of PRKN proteins on mitochondria in siRab7 treated oocytes. Red, PRKN; Green, TOMM20; Pink, DNA was labeled with Hoechst 33342. Oocytes with PRKN translocations were compared between control and treated group. n = 20 oocytes in each group. Bar: 20 μm. (F) The blockage of mitophagic flux after siRab7 treatment. mt-Keima mRNAs were microinjected with siCtrl or siRab7. Images of oocytes were collected at fluorescent emission 550 nm (red) and 440 nm (green), respectively. The ratio (550;440 per oocyte) of fluorescent density was used as an index of mitophagic activity. n = 15 oocytes in each group. Bar: 20 μm. (G) Western Blots of RAB7, LAMP1, PRKN and PINK1 in siCtrl and siRab7 treated groups. The expression of TUBB was used as internal control. The relative intensity of each protein was shown as compared to the expression of TUBB. *, P < 0.05; **, P < 0.01 compared to the control group.

Article Snippet: Primary and secondary antibodies were obtained from the following commercial sources: anti-RAB7A antibody (Cell Signaling Technology, 95,746), anti-PRKN antibody (ZEN BIO, 381,626), anti-ATG9A antibody (ZEN BIO, 381,260), anti-LAMP1 antibody (DSHB, 1D4B), anti-POU5F1/OCT4 antibody (Abcam, ab25630) and anti-TOMM20 antibody (Santa Cruz Biotechnology, sc-17764), anti-TUBB (Cell Signaling Technology, 2146).

Techniques: Immunofluorescence, Labeling, Staining, Double Staining, Translocation Assay, Activity Assay, Western Blot, Expressing

Journal: Autophagy

Article Title: RAB7 activity is required for the regulation of mitophagy in oocyte meiosis and oocyte quality control during ovarian aging

doi: 10.1080/15548627.2021.1946739

Figure Lengend Snippet: CCCP treatment induced PRKN-dependent ubiquitination of RAB7 and MON1-CCZ1 complex in oocytes and 293 T cells. (A-E) Increased ubiquitination of RAB7, CCZ1 and MON1 after CCCP treatment. GV oocytes (n = 800) were treated with 1 µM CCCP for 0.5 h with or without the co-treatment of the proteasome inhibitor, MG132 10 µM. Input shows the protein levels of PRKN, MON1, CCZ1, RAB7 and UB in each group and TUBB was used as the internal control (A and B). The proteins were then immunoprecipitated with RAB7 (C), MON1 (D) and CCZ1 (E) respectively. Their ubiquitin levels were checked by the UB antibody. (F-K) In vitro ubiquitination assay of RAB7, CCZ1 and MON1 in 293 T cells. Plasmids of GFP-Rab7-FLAG, GFP-Mon1-FLAG, GFP-Ccz1-FLAG (2 µg/105 cells) were transfected into 293 T cells with Ub-MYC (2 µg/105 cells) and different does of mCherry-Prkn-HA (2 µg or 4 µg/105 cells) plasmids, respectively. The 293 T cells were pre-treated with 10 μM MG132 for 6 h. After 48 h of transfection, the transfected 293 T cells were then treated with or without CCCP for 2 h. Western blots of inputs showed the protein levels of exogenous UB, PRKN, RAB7, MON1 and CCZ1 with MYC, HA and FLAG antibodies. CCCP treatment induced significant decrease of RAB7 (F), CCZ1 (G) and MON1 (H) expressions in a PRKN-does dependent manner. Proteins in each group were immunoprecipitated with FLAG antibodies respectively and their ubiquitin levels were checked with MYC antibody. PRKN-dependent ubiquitination of RAB7 (I), CCZ1 (J) and MON1 (K) was only seen after CCCP treatment.

Article Snippet: Primary and secondary antibodies were obtained from the following commercial sources: anti-RAB7A antibody (Cell Signaling Technology, 95,746), anti-PRKN antibody (ZEN BIO, 381,626), anti-ATG9A antibody (ZEN BIO, 381,260), anti-LAMP1 antibody (DSHB, 1D4B), anti-POU5F1/OCT4 antibody (Abcam, ab25630) and anti-TOMM20 antibody (Santa Cruz Biotechnology, sc-17764), anti-TUBB (Cell Signaling Technology, 2146).

Techniques: Immunoprecipitation, In Vitro, Ubiquitin Assay, Transfection, Western Blot

Journal: Autophagy

Article Title: RAB7 activity is required for the regulation of mitophagy in oocyte meiosis and oocyte quality control during ovarian aging

doi: 10.1080/15548627.2021.1946739

Figure Lengend Snippet: The activation of PRKN-mediated mitophagy pathway and the blockage of mitophagic flux in aged GV oocytes. GV oocytes were collected from PMSG primed mice at 4 W, 6 M, 8 M and 12 M. (A) Western blots of RAB7, PRKN and PINK1 protein levels in oocytes collected from mice at different ages. The expression of TUBB was used as the internal control. Relative density of each protein was shown as compared with TUBB. (B) Co-staining of PRKN and RAB7 in oocytes at different meiotic stages. Green, RAB7; Red, PRKN; Pink, DNA labeled with Hochest 33342. (C) Increased translocation of active RAB7 on mitochondria in aged oocytes. GV oocytes were collected from 4 W and 12 M mice and microinjected with mCherry-Rab7Q67L mRNA (red) in M2 medium containing 2 μM milrinone for 2 h. Mitochondria was labeled with MitoTracker staining (green) and DNA was counterstained with Hochest 33342 (blue). (D) Oocytes with translocation of active RAB7 on mitochondria were counted and shown as percentage of accumulation. n = 20 oocytes in each group. (E) Decreased mitophagy activity in aged GV oocytes. GV oocytes collected from 4 W and 12 M mice were microinjected with mt-Keima mRNAs and hold in 2 μM milrinone for 8–10 h. Images of oocytes were collected at fluorescent emission 550 nm (red) and 440 nm (green), respectively. (F) The ratio of 550:440 per oocyte was used as an index to reflect mitophagic activity. n = 15 oocytes in each group. (G) ATP levels in oocytes collected from different ages. Increased ATP levels was observed when 12 M oocytes were incubated with 1 µM ML098 for 0.5 h. Bars: 20 µm. *, P < 0.05; **, P < 0.01.

Article Snippet: Primary and secondary antibodies were obtained from the following commercial sources: anti-RAB7A antibody (Cell Signaling Technology, 95,746), anti-PRKN antibody (ZEN BIO, 381,626), anti-ATG9A antibody (ZEN BIO, 381,260), anti-LAMP1 antibody (DSHB, 1D4B), anti-POU5F1/OCT4 antibody (Abcam, ab25630) and anti-TOMM20 antibody (Santa Cruz Biotechnology, sc-17764), anti-TUBB (Cell Signaling Technology, 2146).

Techniques: Activation Assay, Western Blot, Expressing, Staining, Labeling, Translocation Assay, Activity Assay, Incubation

Journal: Autophagy

Article Title: RAB7 activity is required for the regulation of mitophagy in oocyte meiosis and oocyte quality control during ovarian aging

doi: 10.1080/15548627.2021.1946739

Figure Lengend Snippet: In vivo treatment of ML098 mitigated age-related mitochondria damage and improved oocyte quality. Female mice at 10 M of age were i.p. injected 200 µl RAB7 activator ML098 (1 µM) every other day for one month. Mice with saline injection were served as the control group (Ctrl). (A) ROS staining of MII oocytes collected from control and ML098 treated mice after superovulation. ROS levels were reflected by fluorescence intensity in each group. n = 10 oocytes in each group. (B) Early embryonic development after IVF. Mature oocytes were collected from control (n = 44) and ML098 (n = 52) treated mice after superovulation. Early embryonic development was evaluated at 24 h, 48 h, 72 h and 96 h which represented 2-cell, 4-cell, morula and blastocyst stage respectively. (C) TUNEL and POU5F1 staining in blastocysts collected from control and ML098 treated groups. Left panel, TUNEL staining. Right panel, POU5F1 staining to label inner cell mass of the blastocyst. Blue, DNA labeled with Hochest 33342. (D) TUNEL-positive cells (green) in each blastocyst were counted. (E) Inner cell numbers and total cell numbers were counted in each blastocyst. n = 10 blastocysts in each group. (F) Fertility test after in vivo ML098 treatment. Mice (n = 8) in control and ML098 treated mice were mated with fertile male mice to follow the first delivery of pups. Average pups delivered per female were shown. All bars: 20 µm. *, P < 0.05; **, P < 0.01. (G) A schematic diagram depicting the role of RAB7 in regulating PRKN-mediated mitophagy pathway in oocyte meiosis and oocyte quality control during aging. Left panel showed RAB7 activity is required to maintain mitophagy in an inactive state for the progression of meiosis. RAB7 activity is also essential for the auto/mitophagosome formation in oocytes. Right panel demonstrates the induction of PRKN-mediated mitophagy pathway by the accumulation of mitochondria damage in oocytes during aging. The activation of PRKN-mediated mitophagy pathway in aged oocytes is manifested by increased PRKN protein levels and translocations of PRKN on mitochondria outer membrane. PRKN then recruits RAB7 and its GEF complex MON1-CCZ1 to mitochondria outer membrane and ubiquitinates them for degradation through the ubiquitin-proteasome system. The significant decrease of RAB7 activity further induces the failure of auto/mitophagosome formation and the accumulation of damaged mitochondria in cytoplasm which in turn results in meiotic abnormalities and the deterioration of oocyte quality.

Article Snippet: Primary and secondary antibodies were obtained from the following commercial sources: anti-RAB7A antibody (Cell Signaling Technology, 95,746), anti-PRKN antibody (ZEN BIO, 381,626), anti-ATG9A antibody (ZEN BIO, 381,260), anti-LAMP1 antibody (DSHB, 1D4B), anti-POU5F1/OCT4 antibody (Abcam, ab25630) and anti-TOMM20 antibody (Santa Cruz Biotechnology, sc-17764), anti-TUBB (Cell Signaling Technology, 2146).

Techniques: In Vivo, Injection, Staining, Fluorescence, TUNEL Assay, Labeling, Activity Assay, Activation Assay

Journal: Cells

Article Title: BNIP3-Dependent Mitophagy via PGC1α Promotes Cartilage Degradation

doi: 10.3390/cells10071839

Figure Lengend Snippet: PGC1α suppression is associated with BNIP3-induced mitophagy during OA pathogenesis. ( A ) Representative images of Keima-Red with or without IL-1β in iMACs ( n = 4; Scale bars, 100 μm). Results are representative of at least five independent experiments. ( B ) Representative images of LC3-GFP and Keima-Red with the introduction of siPgc1a into iMACs ( n = 5; Scale bars, 100 μm). The results are representative of at least four independent experiments. ( C ) The mitochondrial protein expression level of BNIP3, PARK2 with the introduction of siPgc1a into iMACs. TOMM20 was used for loading control. Each protein level was measured using ImageJ software and normalized by TOMM20 expression level and indicated by a fold change. ( D ) The transcription level of mitophagy genes was analyzed using qRT-PCR ( n = 3). ( E ) Representative images of LC3-GFP and MitoTracker with the introduction of Bnip3 into iMACs ( n = 5; Scale bars, 20 μm). Results are representative of at least five independent experiments. ( F ) Mitochondria membrane potential level was analyzed using MUSE Cell Analyzer ( n = 3). Values were expressed as means ± s.d. An unpaired t -test or one-way ANOVA was used for statistical analysis. * p ≤ 0.05, n.s., non-significant, *** p < 0.001, **** p < 0.0001.

Article Snippet: The following antibodies were used for western blotting; rabbit anti-PGC1α (Abcam, #Ab54481, 1:1000 dilution), rabbit anti-autophagy related 12 (ATG12) (Cell Signaling, #4180, 1:1000 dilution), rabbit anti-Beclin 1 (Cell Signaling, #3495, 1:1000 dilution), rabbit anti-autophagy marker light chain 3 (LC3)B (Cell Signaling, #3868, 1:1000 dilution), rabbit anti-Parkinson’s disease 2 (PARK2) (MyBioSource, San Deigo, CA, USA; #MBS178284, 1:1000 dilution), rabbit anti-GAPDH (Bioworld Technology, St Louis Park, MN, USA; #AP0066, 1:5000 dilution), rabbit anti-translocase of outer mitochondrial membrane 20 (TOMM20) (Abcam, #ab186734, 1:1000 dilution), HRP-conjugated goat anti-rabbit IgG (Enzo Life Sciences, #ADI-SAB-300).

Techniques: Expressing, Control, Software, Quantitative RT-PCR, Membrane

Journal: Cells

Article Title: BNIP3-Dependent Mitophagy via PGC1α Promotes Cartilage Degradation

doi: 10.3390/cells10071839

Figure Lengend Snippet: miR-126-5p dysregulates the homeostasis of cartilage matrix. ( A ) Efficiency of miR-126-5p mimic and inhibitor was confirmed by real-time PCR using iMACs. Scramble-miR was used as control (Con-miR). ( B ) iMACs seeded with the density of 1 × 10 4 /24 well culture dish were transfected with miR-126-5p mimic or inhibitor, stained with Alcian blue (left panel) and quantified based on absorbance at 600 nm (right panel). The results shown are representative of at least three independent experiments. ( C ) The transcription level of Pgc1a , Acan , Col2a1 , and Comp were analyzed using qRT-PCR ( n = 3). ( D ) Representative images of LC3-GFP and MitoTracker with the introduction of miR-126-5p mimic into iMACs ( n = 5; Scale bars, 20 μm). Results are representative of at least five independent experiments. ( E ) Transcription level of Pgc1a , Bnip3 , Pink1 , and Prkn were analyzed using qRT-PCR ( n = 3). Values were expressed as means ± s.d. An unpaired t -test or one-way ANOVA was used for statistical analysis. * p ≤ 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The following antibodies were used for western blotting; rabbit anti-PGC1α (Abcam, #Ab54481, 1:1000 dilution), rabbit anti-autophagy related 12 (ATG12) (Cell Signaling, #4180, 1:1000 dilution), rabbit anti-Beclin 1 (Cell Signaling, #3495, 1:1000 dilution), rabbit anti-autophagy marker light chain 3 (LC3)B (Cell Signaling, #3868, 1:1000 dilution), rabbit anti-Parkinson’s disease 2 (PARK2) (MyBioSource, San Deigo, CA, USA; #MBS178284, 1:1000 dilution), rabbit anti-GAPDH (Bioworld Technology, St Louis Park, MN, USA; #AP0066, 1:5000 dilution), rabbit anti-translocase of outer mitochondrial membrane 20 (TOMM20) (Abcam, #ab186734, 1:1000 dilution), HRP-conjugated goat anti-rabbit IgG (Enzo Life Sciences, #ADI-SAB-300).

Techniques: Real-time Polymerase Chain Reaction, Control, Transfection, Staining, Quantitative RT-PCR