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

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