Journal: Heliyon

Article Title: Shiga toxin 2 A-subunit induces mitochondrial damage, mitophagy and apoptosis via the interaction of Tom20 in Caco-2 cells

doi: 10.1016/j.heliyon.2023.e20012

Figure Lengend Snippet: PINK1 and Parkin mediate Stx2-induced mitophagy in Caco-2 cells. (A–D) Caco-2 cells were transfected with 1 μg/ml Stx2A plasmids for 12, 24 and 48 h. Representative Western blot of PINK1 and Parkin in whole cells (A) as well as PINK1 and Parkin in mitochondria (B), and representative images of PINK1 and MitoTracker immunofluorescence (C) or Parkin and MitoTracker immunofluorescence (D). (E, F) Caco-2 cells were cotransfected with 1 μg/ml Stx2A plasmids and siRNAs, including negative control siRNA (siNC), PINK1 siRNA (siPINK1), or Parkin siRNA (siParkin). Representative images of LC3B (green) and Parkin (red) immunofluorescence (E). Scale bar = 5 μm. The protein expression of PINK1, Parkin, LC3 II/I, Beclin1, Tom20 and SQSTM1 was detected by Western blot analysis (F). n = 3. * P < 0.05.

Article Snippet: The following antibodies were utilized in the present study: MnSOD (ab137037), cytochrome c (ab13575) and Parkin (ab77924, ab15494) from Abcam (Shanghai, China); SQSTM1 (88588), LC3B (83506), COX IV (4850), Cleaved-Caspase-3 (9661), PINK1 (6946), Tom20 (42406), Cleaved-Caspase-9 (20750), Bax (2772), GAPDH (2118), Beclin-1 (3738) and Hsp60 (12165) from Cell Signaling Technology (Shanghai, China); Bcl-2 (12789), Bax (60267) and VDAC1 (66345) from Proteintech (Wuhan, China); and PINK1 (BC100–494) and 8-OHdG (NB110-96878) from Novas Biologicals (Shanghai, China).

Techniques: Transfection, Western Blot, Immunofluorescence, Negative Control, Expressing

Journal: Heliyon

Article Title: Shiga toxin 2 A-subunit induces mitochondrial damage, mitophagy and apoptosis via the interaction of Tom20 in Caco-2 cells

doi: 10.1016/j.heliyon.2023.e20012

Figure Lengend Snippet: PINK1/Parkin-dependent mitophagy decreases mitochondrial ROS production and apoptosis induced by Stx2A. (A, B) Caco-2 cells were cotransfected with 1 μg/ml Stx2A plasmids and control siRNA, PINK1 siRNA, or Parkin siRNA. Cells were then treated with MitoTEMPO (100 μM) for 4 h followed by additional culture for 24 h. Representative images of mitochondrial ROS (MitoSOX, Red) and 8-OHdG (Green) immunofluorescence (A) as well as flow cytometry analysis of mitochondrial ROS by MitoSOX (B) in Caco-2 cells. Scale bar = 5 μm. (C) Representative Western blot analysis of PINK1, Parkin, LC3 II/I, Beclin1, Tom20 and SQSTM1 after transfection with 1 μg/ml Stx2A plasmids for 24 h followed by treatment with MitoTEMPO (100 μM) for 4 h. (D, E) Caco-2 cells were treated with or without MitoTEMPO after silencing PINK1 or Parkin and transfection with Stx2A plasmids. Western blot analysis (D) of Cleaved-Caspase-3, Cleaved-Caspase-9, Bax and Bcl-2. Representative images and quantification of cell apoptosis by flow cytometry (E). n = 3. * P < 0.05.

Article Snippet: The following antibodies were utilized in the present study: MnSOD (ab137037), cytochrome c (ab13575) and Parkin (ab77924, ab15494) from Abcam (Shanghai, China); SQSTM1 (88588), LC3B (83506), COX IV (4850), Cleaved-Caspase-3 (9661), PINK1 (6946), Tom20 (42406), Cleaved-Caspase-9 (20750), Bax (2772), GAPDH (2118), Beclin-1 (3738) and Hsp60 (12165) from Cell Signaling Technology (Shanghai, China); Bcl-2 (12789), Bax (60267) and VDAC1 (66345) from Proteintech (Wuhan, China); and PINK1 (BC100–494) and 8-OHdG (NB110-96878) from Novas Biologicals (Shanghai, China).

Techniques: Control, Immunofluorescence, Flow Cytometry, Western Blot, Transfection

Journal: Heliyon

Article Title: Shiga toxin 2 A-subunit induces mitochondrial damage, mitophagy and apoptosis via the interaction of Tom20 in Caco-2 cells

doi: 10.1016/j.heliyon.2023.e20012

Figure Lengend Snippet: Stx2A induces mitochondrial damage, mitophagy and apoptosis through Tom20. (A–E) Caco-2 cells were cotransfected with Tom20 shRNA and 1 μg/ml Stx2A plasmids for 24 h. The protein expression levels of MnSOD and COX Ⅳ were determined by Western blot analysis (A). Representative images and quantification of mitochondrial superoxide levels (B) and MMP (C) by flow cytometry. Representative Western blot images (D) of PINK1, Parkin, LC3 II/I, Beclin1 and SQSTM1. Representative images of mitophagosomes obtained by LC3B (green) and Parkin (red) (E) immunofluorescence. Scale bar = 5 μm. (F) Caco-2 cells were transfected with Stx2A plasmids for 24 h in the presence or absence of 0.2 μM bafilomycin A1 or 5 μM lactacystin. Representative Western blot of LC3BII and Tom20. (G, H) Caco-2 cells were transfected with Tom20 shRNA and 1 μg/ml Stx2A plasmids for 24 h. Immunoblot analysis (G) of Cleaved-Caspase-3, Cleaved-Caspase-9, Bax, cytochrome c (Cyto C) and Bcl-2 in Caco-2 cells. Detection of apoptosis using the annexin V-FITC/PI kit by flow cytometry (H). n = 3. * P < 0.05.

Article Snippet: The following antibodies were utilized in the present study: MnSOD (ab137037), cytochrome c (ab13575) and Parkin (ab77924, ab15494) from Abcam (Shanghai, China); SQSTM1 (88588), LC3B (83506), COX IV (4850), Cleaved-Caspase-3 (9661), PINK1 (6946), Tom20 (42406), Cleaved-Caspase-9 (20750), Bax (2772), GAPDH (2118), Beclin-1 (3738) and Hsp60 (12165) from Cell Signaling Technology (Shanghai, China); Bcl-2 (12789), Bax (60267) and VDAC1 (66345) from Proteintech (Wuhan, China); and PINK1 (BC100–494) and 8-OHdG (NB110-96878) from Novas Biologicals (Shanghai, China).

Techniques: shRNA, Expressing, Western Blot, Flow Cytometry, Immunofluorescence, Transfection

Journal: Heliyon

Article Title: Shiga toxin 2 A-subunit induces mitochondrial damage, mitophagy and apoptosis via the interaction of Tom20 in Caco-2 cells

doi: 10.1016/j.heliyon.2023.e20012

Figure Lengend Snippet: Tom20-induced translocation of Bax to mitochondria contributes to mitochondrial damage, mitophagy and apoptosis. (A) Bax (green) translocated to mitochondria after transfection with 1 μg/ml Stx2A plasmids. Hsp60 (red) was localized to mitochondria. Scale bar = 5 μm. (B) Knockdown of Tom20 blocked the translocation of Bax to mitochondria after Stx2A transfection. (C–I) Caco-2 cells were transfected with Bax shRNA and Stx2A plasmids for 24 h. The protein expression levels of MnSOD and COX Ⅳ were determined by Western blot analysis (C). Representative images of mitochondrial superoxide levels (D) and MMP (E) by flow cytometry. Representative Western blot images (F) of PINK1, Parkin, LC3 II/I, Beclin1 and SQSTM1. Representative immunofluorescence images of mitophagosomes (G) with LC3B and Parkin. Western blot analysis (H) of Cleaved-Caspase-3, Cleaved-Caspase-9, Bax, cytochrome c (Cyto C) and Bcl-2 in Caco-2 cells. Apoptosis was detected using an annexin V-FITC/PI kit and flow cytometry (I). n = 3. * P < 0.05.

Article Snippet: The following antibodies were utilized in the present study: MnSOD (ab137037), cytochrome c (ab13575) and Parkin (ab77924, ab15494) from Abcam (Shanghai, China); SQSTM1 (88588), LC3B (83506), COX IV (4850), Cleaved-Caspase-3 (9661), PINK1 (6946), Tom20 (42406), Cleaved-Caspase-9 (20750), Bax (2772), GAPDH (2118), Beclin-1 (3738) and Hsp60 (12165) from Cell Signaling Technology (Shanghai, China); Bcl-2 (12789), Bax (60267) and VDAC1 (66345) from Proteintech (Wuhan, China); and PINK1 (BC100–494) and 8-OHdG (NB110-96878) from Novas Biologicals (Shanghai, China).

Techniques: Translocation Assay, Transfection, Knockdown, shRNA, Expressing, Western Blot, Flow Cytometry, Immunofluorescence

Journal: Heliyon

Article Title: Shiga toxin 2 A-subunit induces mitochondrial damage, mitophagy and apoptosis via the interaction of Tom20 in Caco-2 cells

doi: 10.1016/j.heliyon.2023.e20012

Figure Lengend Snippet: Schematic representation of mitochondrial damage, mitophagy and apoptosis in Caco-2 cells. Stx2A causes the mitochondrial damage of Caco-2 cells, which induces mitophagy and apoptosis activation via the interaction of Tom20 in Caco-2 cells, and PINK1/Parkin–mediated mitophagy ameliorates apoptosis by eliminating damaged mitochondria.

Article Snippet: The following antibodies were utilized in the present study: MnSOD (ab137037), cytochrome c (ab13575) and Parkin (ab77924, ab15494) from Abcam (Shanghai, China); SQSTM1 (88588), LC3B (83506), COX IV (4850), Cleaved-Caspase-3 (9661), PINK1 (6946), Tom20 (42406), Cleaved-Caspase-9 (20750), Bax (2772), GAPDH (2118), Beclin-1 (3738) and Hsp60 (12165) from Cell Signaling Technology (Shanghai, China); Bcl-2 (12789), Bax (60267) and VDAC1 (66345) from Proteintech (Wuhan, China); and PINK1 (BC100–494) and 8-OHdG (NB110-96878) from Novas Biologicals (Shanghai, China).

Techniques: Activation Assay

Journal: Molecules

Article Title: Nujiangexanthone A Inhibits Cervical Cancer Cell Proliferation by Promoting Mitophagy

doi: 10.3390/molecules26102858

Figure Lengend Snippet: Figure 1. NJXA promotes Parkin-dependent mitophagy in HeLa cells. (A) YFP-Parkin HeLa cells were treated with NJXA (20 µM) for 0–24 h, and the distribution of YFP-Parkin was examined by immunofluorescence microscopy. Scale bar = 20 µM. (B) HeLa cells treated with NJXA (20 µM) for 0–24 h were analyzed by immunoblotting for PINK1, PARKIN, TOM20, TIM23, MFN1, and MFN2. GAPDH was served as loading control. The values above the bands indicate the relative 669 band intensities. Data are presented as the means ± SD of three independent experiments.

Article Snippet: The human-specific antibodies used included GAPDH (ab128915) from Abcam; Tom20 (sc-11415), Parkin (sc-133167), and Tim23 (sc-514463) from Santa Cruz; p62 (23214), PINK1 (6946), and LC3 (3868) from Cell Signaling Technology (Boston, MA, USA); and MFN1 (66776-1-Ig) and MFN2 (67487-1-Ig) from Proteintech (Chicago, IL, USA).

Techniques: Microscopy, Western Blot, Control

Journal: Molecules

Article Title: Nujiangexanthone A Inhibits Cervical Cancer Cell Proliferation by Promoting Mitophagy

doi: 10.3390/molecules26102858

Figure Lengend Snippet: Figure 5. Effect of NJXA on HeLa cells is partially reversed by mitophagy inhibition. (A) HeLa-ctr and HeLa-ATG7 cells were treated with NJXA at the indicated concentrations (0–20 µM) for 72 h and evaluated by CCK-8 assay. Data is shown as mean ± SD of three independent experiments, ** p < 0.01, and *** p < 0.001. (B) HeLa-ctr and HeLa-ATG7 cells were treated with NJXA (20 µM) for 0–24 h and analyzed by immunoblotting for ATG7, PINK1, PARKIN, MFN2, LC3, and LAMP1. GAPDH served as the loading control. The values above the bands indicate the relative 669 band intensities. Data are presented as the means ± SEM of three independent experiments. (C) HeLa-ATG7 cells treated with NJXA (20 µM) for 0, 2, and 4 h were collected for mitochondrial membrane potential analysis. (D) YFP-Parkin HeLa cells transfected with ATG7 or control plasmid cells were treated with NJXA (20 µM) for 4 h, fixed, and stained with MFN2, p62, and LAMP1 antibodies. The images were acquired using a fluorescence microscope. Scale bar = 20 µM.

Article Snippet: The human-specific antibodies used included GAPDH (ab128915) from Abcam; Tom20 (sc-11415), Parkin (sc-133167), and Tim23 (sc-514463) from Santa Cruz; p62 (23214), PINK1 (6946), and LC3 (3868) from Cell Signaling Technology (Boston, MA, USA); and MFN1 (66776-1-Ig) and MFN2 (67487-1-Ig) from Proteintech (Chicago, IL, USA).

Techniques: Inhibition, CCK-8 Assay, Western Blot, Control, Membrane, Transfection, Plasmid Preparation, Staining, Microscopy

Journal: Antioxidants

Article Title: 25-Hydroxycholecalciferol Improves Cardiac Metabolic Adaption, Mitochondrial Biogenetics, and Redox Status to Ameliorate Pathological Remodeling and Functional Failure in Obese Chickens

doi: 10.3390/antiox13111426

Figure Lengend Snippet: Effects of dietary 25-hydroxycholecalciferol (25-OH-D3) supplementation on cardiac mitochondrial biogenesis and dynamics of broiler breeder hens provided with restricted (R) or ad libitum (Ad) feed intake. Hearts collected at 28, 33, and 45 weeks were used for relative mitochondrial DNA quantification using total DNA extracts for qRT-PCR with specific primers targeted to mitochondrial (COX1, CYTB, and ND1) and nuclear (GAPDH) genes ( A ). Mitochondrial fractions were used for actual DNA content determination using a fluorescence dye method ( B ). Activation of PGC-1α (PPAR-γ coactivator 1-alpha), PINK1 (PTEN-induced kinase 1), and Parkin expressions for mitophagy were analyzed by Western blotting with nuclear extracts and total cell lysates, respectively ( C ). Results of qRT-PCR and Western blot were normalized to GADPH and β-actin, respectively, and expressed as ratios relative R-hens at 28, 33, or 45 weeks (n = 4) *; significant difference by Ad-feed intake (vs. corresponding R-hens, p < 0.05) +; significant difference by 25-OH-D3 (vs. R- or Ad-hens, p < 0.05). ND1; NADH ubiquinone oxidoreductase chain 1, CYTB; cytochrome b, COX1; cytochrome oxidase 1, GADPH; glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Left ventricle homogenates in RIPA buffer, and nuclear extracts prepared using a commercial kit (Cat. #ab113474, Abcam), were used for Western blot analysis using antibodies cross-reactive to chicken antigens including rabbit anti-Nfr-2 (Nuclear factor erythroid 2-related factor 2, Cat. #ab31163) and goat anti-PGC-1α (PPAR-α coactivator 1-alpha, Cat. #ab106814) antibody from Abcam, and rabbit anti-β-actin (Cat. #4967) antibody from Cell Signaling Technology (Danvers, MA, USA), mouse anti-FECH (ferrochelatase, Cat. #sc-377377), HO-1 (heme oxygenase-1, Cat. #sc-390911), Pink1 (PTEN-induced kinase 1, Cat. #sc-518052), and Parkin (Parkinson juvenile disease protein 2, Cat. #sc-32282) antibody from Santa Cruz (Dallas, TX, USA), GPX4 antibody (Cat. #67763-1-Ig) from Proteintech Group, Inc (Rosemont, IL, USA).

Techniques: Quantitative RT-PCR, Fluorescence, Activation Assay, Western Blot