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 mitophagy receptor BNIP3 is critical for the regulation of metabolic homeostasis and mitochondrial function in the nucleus pulposus cells of the intervertebral disc

doi: 10.1080/15548627.2022.2162245

Figure Lengend Snippet: BNIP3 loss does not result in a compensatory increase in levels and colocalization of other mitophagy receptors. (A, B) Western blot and densitometric quantification of mitophagy receptors BNIP3L, BCL2L13, and FUNDC1 in ShCtrl, Sh Bnip3 #1, and DFP-treated NP cells. (C, D) Immunofluorescence staining BNIP3L and quantification of BNIP3L colocalizing with mitochondria, and (E, F) Immunofluorescence staining FUNDC1 and quantification of FUNDC1 colocalizing with mitochondria. (G, H) Western blot and densitometric quantification of canonical mitophagy pathway protein PRKN in ShCtrl, Sh Bnip3 , and DFP-treated NP cells. Western blot data represent six independent experiments. Colocalization BNIP3L and FUNDC1 with mitochondria was measured from Z-stack images of 100 cells per group. Scale bar: 15 and 4 μm. Statistical significance was determined using a t-test (D, F) or One-way ANOVA (B, H) with Sidaks’s post hoc test as appropriate.

Article Snippet: Primary NP cells from Sprague Dawley (Charles River, strain 400), prkn / park2 KO and WT (Envigo, HsdSage:LE- Park2 em1Sage ) and bnip3 KO-mitoQC and WT-mitoQC reporter mice were isolated and maintained in Dulbecco’s modified Eagle medium (DMEM; Sigma, D6046) and 10% FBS supplemented with antibiotics [ ].

Techniques: Western Blot, Immunofluorescence, Staining

Journal: Cell & bioscience

Article Title: Mitochondria directly donate their membrane to form autophagosomes during a novel mechanism of parkin-associated mitophagy.

doi: 10.1186/2045-3701-4-16

Figure Lengend Snippet: Figure 1 Drug-induced autophagy in ER + breast cancer cells exhibit elevated autophagosome formation and indicate a direct contribution of the mitochondrial membrane in autophagosome membrane development. Electron micrograph images of A. vehicle treated LCC9 breast cancer cells. B. LCC9 cells treated with 100 nM fulvestrant for 72 hours. C. LCC9 cells transfected with estrogen receptor-α shRNA. D. LCC9 cells treated with 10 μM Imatinib for 72 hours. M indicates mitochondria; Av indicates an autophagic vesicle; * indicates mitochondria-autophagosome interaction.

Article Snippet: The following materials were obtained as indicated: Imatinib and ICI 182,780 (Tocris Bioscience, Ellisville, MO); penicillin and Improved Minimal Essential Medium (IMEM; Gibco Invitrogen BRL, Carlsbad, CA); bovine calf charcoal stripped serum (CCS) (Equitech-Bio Inc, Kerrville, TX); Lipofectamine RNAiMax reagent (Invitrogen); Estrogen receptor-α (ER) shRNA (Evrogen, Moscow, Russia); GFP-LC3 (Addgene, Cambridge, MA); EndoTracker Red, Golgi-RFP, MitoTracker-GFP, MitoTracker-RFP (Invitrogen); Cyto-ID Autophagosome detection kit (Enzo Life Sciences); LC3B and parkin antibody (Cell Signaling Technology, Danvers, MA); PINK1 and parkin siRNA (Origene, Rockville, MD). vated autophagosome formation and indicate a direct contribution ment.

Techniques: Membrane, Transfection, shRNA

Journal: Cell & bioscience

Article Title: Mitochondria directly donate their membrane to form autophagosomes during a novel mechanism of parkin-associated mitophagy.

doi: 10.1186/2045-3701-4-16

Figure Lengend Snippet: Figure 2 Mitochondria directly donate membrane material to form autophagosomes. Electron micrograph images of A. vehicle treated LCC9 breast cancer cells. B. LCC9 cells treated with 100 nM fulvestrant for 72 hours. C. LCC9 cells transfected with estrogen receptor-α shRNA. D. LCC9 cells treated with 10 μM Imatinib for 72 hours. M indicates mitochondria; Av indicates an autophagic vesicle; * indicates mitochondria-autophagosome interaction.

Article Snippet: The following materials were obtained as indicated: Imatinib and ICI 182,780 (Tocris Bioscience, Ellisville, MO); penicillin and Improved Minimal Essential Medium (IMEM; Gibco Invitrogen BRL, Carlsbad, CA); bovine calf charcoal stripped serum (CCS) (Equitech-Bio Inc, Kerrville, TX); Lipofectamine RNAiMax reagent (Invitrogen); Estrogen receptor-α (ER) shRNA (Evrogen, Moscow, Russia); GFP-LC3 (Addgene, Cambridge, MA); EndoTracker Red, Golgi-RFP, MitoTracker-GFP, MitoTracker-RFP (Invitrogen); Cyto-ID Autophagosome detection kit (Enzo Life Sciences); LC3B and parkin antibody (Cell Signaling Technology, Danvers, MA); PINK1 and parkin siRNA (Origene, Rockville, MD). vated autophagosome formation and indicate a direct contribution ment.

Techniques: Membrane, Transfection, shRNA

Journal: Cell & bioscience

Article Title: Mitochondria directly donate their membrane to form autophagosomes during a novel mechanism of parkin-associated mitophagy.

doi: 10.1186/2045-3701-4-16

Figure Lengend Snippet: Figure 4 Vesicles forming from mitochondria are autophagosomes. A. LCC9 cells were transfected with GFP-LC3, treated with 0.1% ethanol vehicle, 500 nM ICI, ERα shRNA, or 10 μM Imatinib for 24 hours and counterstained with DAPI. Confocal microscopy was used to determine LC3-positive puncta formation. B. LCC9 cells were treated with 0.1% ethanol vehicle, 100 nM ICI, ERα shRNA, or 10 μM Imatinib for 72 hours and protein isolated. Western blot hybridization was used to determine LC3-I/LC3-II and p62 levels. C. LCC9 cells were treated with vehicle, 500 nM ICI, serum starvation, 2 ng/mL tunicamycin, 10 μM Imatinib, or transfected with ATG7 siRNA, PARK2 (parkin) siRNA, or ERα shRNA for 24 hours. Autophagosome (modified monodansylcadaverine) and mitochondria (MitoTracker-GFP) fold change was determined by flow cytometry. D. LC3-immunogold EM of LCC9 cells. M indicates mitochondria; Av indicates an autophagic vesicle; * indicates mitochondria-autophagosome interaction; arrows indicate LC3-immunogold particles. E. Mitochondria were counted and represented as percent of mitochondria either labeled with LC3-immunogold or unlabeled. n = 3-4, *p < 0.05.

Article Snippet: The following materials were obtained as indicated: Imatinib and ICI 182,780 (Tocris Bioscience, Ellisville, MO); penicillin and Improved Minimal Essential Medium (IMEM; Gibco Invitrogen BRL, Carlsbad, CA); bovine calf charcoal stripped serum (CCS) (Equitech-Bio Inc, Kerrville, TX); Lipofectamine RNAiMax reagent (Invitrogen); Estrogen receptor-α (ER) shRNA (Evrogen, Moscow, Russia); GFP-LC3 (Addgene, Cambridge, MA); EndoTracker Red, Golgi-RFP, MitoTracker-GFP, MitoTracker-RFP (Invitrogen); Cyto-ID Autophagosome detection kit (Enzo Life Sciences); LC3B and parkin antibody (Cell Signaling Technology, Danvers, MA); PINK1 and parkin siRNA (Origene, Rockville, MD). vated autophagosome formation and indicate a direct contribution ment.

Techniques: Transfection, shRNA, Confocal Microscopy, Isolation, Western Blot, Hybridization, Modification, Flow Cytometry, Labeling