Journal: Molecular Neurodegeneration

Article Title: A partial Drp1 knockout improves autophagy flux independent of mitochondrial function

doi: 10.1186/s13024-024-00708-w

Figure Lengend Snippet: Drp1 inhibition protects against impaired autophagy and protein aggregation induced by Mn in N27 dopaminergic neuronal cells. Stable N27 cells expressing inducible human wild-type α-synuclein were co-transfected with siRNA-Drp1 (KD) and LC3-mcherry (due to low endogenous LC3 levels in this cell type) for 24 h. ( A ) Cells were then treated for another 24 h with MnCl2 (125 µM) or vehicle controls (Con 1 & Con 2), in the presence or absence of PonA (20 µM) to induce α-synuclein expression. ( B ), Following confocal imaging, the number of green and red vesicles, representing respectively p62 and LC3 puncta was quantified using Imaris image analysis software. Data represent mean ± SEM ( n = 3 independent experiments, with at least 15 cells for each condition). Kruskal-Wallis ANOVA was used to compare between groups with Dunn’s post-hoc test. Scale bar 20 μm. ( C ), Representative confocal images showing total α-synuclein immunostaining (green) with or without Proteinase-K (PK) treatment. ( D ), N27 cells were transfected with siRNA-Drp1 (10nM) for 24 h, followed by α-synuclein overexpression induction (PonA20 µM), with and without Mn (125 µM) for 48 h. After post-fixation, cells were incubated with PK and then incubated with an antibody that detects α-synuclein (Millipore, AB5038). ( E ) Imaris was used to quantify PK-resistant α-synuclein-positive puncta. The number of aggregates was normalized to cytoplasmic volume. Data are shown as mean ± SEM ( n = 3 independent experiments, with a minimum of 40 cells per group). Scale bar 20 μm

Article Snippet: The primary antibodies used were rabbit anti-Atg5 (1:100, cat. no. NB110-53818, Novus Biologicals, Bio-Techne), rabbit anti-Drp1 (1:100, cat. no. NB110-55237, Novus Biologicals, Bio-Techne) and rabbit anti-p62 (1:50, cat. no. NBP1-48320, Novus Biologicals, Bio-Techne).

Techniques: Inhibition, Expressing, Transfection, Imaging, Software, Immunostaining, Over Expression, Incubation

Journal: Molecular Neurodegeneration

Article Title: A partial Drp1 knockout improves autophagy flux independent of mitochondrial function

doi: 10.1186/s13024-024-00708-w

Figure Lengend Snippet: Drp1 +/- mice is protective against autophagy impairment induced by Mn. ( A ) Representative images of the coronal mouse midbrain section (20 μm) co-immunostained for DA neurons (TH, red) in the SNpc and GABA neurons (GAD67, green) in the SNpr. Both of these brain regions were removed by laser microdissection for immunoblotting of p62 (top panels) in DA neurons ( B ) and GABA neurons ( C ). Total proteins per lane (bottom panels) were used as loading control. ( D ) Quantified levels of p62 were significantly increased in DA neurons ( P = 0.0013), but not in the GABA neurons ( P = 0.5457), of the Mn-treated WT mice. Mn did not significantly increase p62 in DA neurons of the Drp1 +/− mice ( P = 0.8660) No significant baseline level differences between the two genotypes were observed ( P = 0.5664 for TH neurons and P = 0.7675 for GABA neurons. Data represent mean ± SEM, n = 5 for WT control, n = 6 for other groups), two-way ANOVA followed by Tukey post-hoc test. ( E ) Representative confocal images of mitochondrial morphology of DA neurons after TOM20 immunostaining (upper panels), then skeletonized (middle panels) for subsequent analysis. Scale bar 20 μm. ( F, G ) Various parameters of mitochondrial morphology were quantified using Fiji MiNA plugin. Data represents mean ± SEM ( n = 6 mice per group), analyzed by one-way ANOVA, followed by Tukey post-hoc test

Article Snippet: The primary antibodies used were rabbit anti-Atg5 (1:100, cat. no. NB110-53818, Novus Biologicals, Bio-Techne), rabbit anti-Drp1 (1:100, cat. no. NB110-55237, Novus Biologicals, Bio-Techne) and rabbit anti-p62 (1:50, cat. no. NBP1-48320, Novus Biologicals, Bio-Techne).

Techniques: Laser Capture Microdissection, Western Blot, Control, Immunostaining

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Hypoxia-Inducible Factor 1-α (HIF-1α) Induces Apoptosis of Human Uterosacral Ligament Fibroblasts Through the Death Receptor and Mitochondrial Pathways

doi: 10.12659/MSM.913384

Figure Lengend Snippet: Expression of death receptor-associated apoptotic markers in the cobalt chloride (CoCl 2 )-treated human uterosacral ligament fibroblasts (hUSLFs). ( A ) Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), Fas, decoy receptor 2 (DcR2), cellular FLICE inhibitory protein (c-FLIP), death receptor 5 (DR5), and cleaved caspase-8 protein levels by Western blot analysis in hUSLFs treated with the chemical inducer of hypoxia, cobalt chloride (CoCl 2 ), at 48 h. ( B ) Quantitative analysis of Fas, TRAIL, DcR2, c-FLIP, and DR5 expression standardized to β-actin. ( C ) Caspase-8 activity measured in hUSLFs treated with CoCl 2 for 48 h. ( D ) After 48 h, the effects of CoCl 2 on the levels of lactate dehydrogenase (LDH) in hUSLFs were measured. ( E ) Results of the MTT assay for cell viability following treatment with CoCl 2 for 48 h with or without the presence of 50 μM of the caspase-8 inhibitor, Z-IETD-FMK. * P <0.05, ** P <0.01. Con – control. C-cas-8 – cleaved caspase-8.

Article Snippet: After pretreated with and without 50 μM of the caspase-8 inhibitor, Z-IETD-FMK (MedChem Express, Monmouth Junction, NJ, USA), and treatment with and without 100 μM of the caspase-9 inhibitor, Z-LEHD-FMK (KeyGen Biotech Co. Ltd., Nanjing, China) for 2 h, cells were treated with CoCl 2 48 h later.

Techniques: Expressing, Western Blot, Activity Assay, MTT Assay, Control

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Hypoxia-Inducible Factor 1-α (HIF-1α) Induces Apoptosis of Human Uterosacral Ligament Fibroblasts Through the Death Receptor and Mitochondrial Pathways

doi: 10.12659/MSM.913384

Figure Lengend Snippet: Expression of mitochondrial-associated apoptotic markers in the cobalt chloride (CoCl 2 )-treated human uterosacral ligament fibroblasts (hUSLFs). ( A ) Bcl-2, Bax, cytochrome C, Bcl-2 interacting protein 3 (BNIP3), cleaved caspase-3, and cleaved caspase-9 protein levels were analyzed by Western blot in human uterosacral ligament fibroblasts (hUSLFs) that were treated with the chemical inducer of hypoxia, cobalt chloride (CoCl 2 ) at 48 h. ( B ) Quantitative analysis of Bcl-2/Bax, cytochrome C, BNIP3, cleaved caspase-3, and cleaved caspase-9 protein expression standardized to β-actin. ( C ) Caspase-3, and caspase-9 activity measured in hUSLFs that were treated with CoCl 2 for 48 h. ( D ) Results of the MTT assay for cell viability following treatment with CoCl 2 for 48 h, which were also exposed to 100 μM of the caspase-9 inhibitor, Z-LEHD-FMK, with or without the presence of 50 μM of the caspase-8 inhibitor, Z-IETD-FMK. ( E ) Photomicrograph of JC-1 fluorescence mitochondrial imaging used to study the change in mitochondrial membrane potential in the different groups. ( F ) The relative mitochondrial membrane potential level was evaluated by comparing the red fluorescence cells to green fluorescence cells in the groups. * P <0.05, ** P <0.01. Scale bar=100 μm. Con – control; C-cas-3 – cleaved caspase-3; C-cas-9 – cleaved caspase-9; Cyto c – cytochrome C.

Article Snippet: After pretreated with and without 50 μM of the caspase-8 inhibitor, Z-IETD-FMK (MedChem Express, Monmouth Junction, NJ, USA), and treatment with and without 100 μM of the caspase-9 inhibitor, Z-LEHD-FMK (KeyGen Biotech Co. Ltd., Nanjing, China) for 2 h, cells were treated with CoCl 2 48 h later.

Techniques: Expressing, Western Blot, Activity Assay, MTT Assay, Fluorescence, Imaging, Membrane, Control

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Hypoxia-Inducible Factor 1-α (HIF-1α) Induces Apoptosis of Human Uterosacral Ligament Fibroblasts Through the Death Receptor and Mitochondrial Pathways

doi: 10.12659/MSM.913384

Figure Lengend Snippet: Knockdown of hypoxia-inducible factor-1α (HIF-1α) prevented cobalt chloride (CoCl 2 )-induced apoptosis in human uterosacral ligament fibroblasts (hUSLFs) by normalizing the expression levels of apoptosis-associated markers, as shown by Western blot of cells with or without HIF-1α knockdown treated with CoCl 2 for 48 h. ( A ) Western blot results for the expression of hypoxia-inducible factor-1α (HIF-1α) and collagen type I alpha 1 (COL1A1). ( B ) Western blot results show the relative band intensities of HIF-1α and COL1A1. ( C ) Flow cytometry shows cell apoptosis by after double-staining with Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI). ( D ) Flow cytometry shows the cell apoptosis rate. ( E ) Western blot results show the relative band intensities of death receptor-associated apoptotic markers. ( F ) Western blot results show the relative band intensities of death receptor-associated apoptotic markers. ( G ) Western blot results show the relative band intensities of mitochondrial-associated apoptotic markers. ( H ) Western blot results show the relative band intensities mitochondrial-associated apoptotic markers. * P <0.05, ** P <0.01, vs. control; # P <0.05, ## P <0.01, ### P <0.001, vs. CoCl 2 + NC. Con – control; C-cas-3 – cleaved caspase-3; C-cas-8 – cleaved caspase-8; C-cas-9 – cleaved caspase-9; Cyto-c – cytochrome C.

Article Snippet: After pretreated with and without 50 μM of the caspase-8 inhibitor, Z-IETD-FMK (MedChem Express, Monmouth Junction, NJ, USA), and treatment with and without 100 μM of the caspase-9 inhibitor, Z-LEHD-FMK (KeyGen Biotech Co. Ltd., Nanjing, China) for 2 h, cells were treated with CoCl 2 48 h later.

Techniques: Knockdown, Expressing, Western Blot, Flow Cytometry, Double Staining, Control

Journal: Stem Cell Research & Therapy

Article Title: hucMSC-derived exosomes attenuate colitis by regulating macrophage pyroptosis via the miR-378a-5p/NLRP3 axis

doi: 10.1186/s13287-021-02492-6

Figure Lengend Snippet: hucMSC-derived exosomes reduce cell pyroptosis by decreasing the activation of NLRP3 inflammasomes both in vivo and in vitro. a IHC analysis of Caspase-1 p45 and Caspase-1 p20 expression in mouse colon tissues (200×, scale bar = 50 μm). b Western blot analysis of GSDMD protein levels in mouse colon tissues and the grayscale scanning analysis. c CCK8 assay analysis of THP-1 cell viability. d CCK8 assay analysis of MPM viability. e LDH activity analysis of THP-1 cell supernatant. f LDH activity analysis of MPM supernatant. g Identification of PI-positive THP-1 cells by flow cytometry. h Western blot analysis of the protein expression level of GSDMD and cleaved GSDMD fragment in both THP-1 cell lysate and MPM lysate and the grayscale scanning analysis. i Imaging assay of pyroptosis in MPMs treated as indicated. # P < 0.05, ## P < 0.01, ### P < 0.001 vs Ctrl by ANOVA; * P < 0.05, ** P < 0.01, *** P < 0.001 vs LPS + NIG by ANOVA

Article Snippet: Primary antibodies such as NLRP3 (1:100; Novus, CO, USA), IL-1β (1:100; Cell Signaling Technology, MA, USA), Caspase-1 p45 (1:100; Proteintech Group, Chicago, IL, USA), and Caspase-1 p20 (1:50; Santa Cruz Biotechnology, CA, USA) were added for overnight incubation at 4 °C, followed by the secondary antibody (Wuhan Boster Biological Technology, Wuhan, China) at 37 °C for 30 min. Then, StreptAvidin Biotin Complex (SABC) was added and incubated at 37 °C for 30 min.

Techniques: Derivative Assay, Activation Assay, In Vivo, In Vitro, Expressing, Western Blot, CCK-8 Assay, Activity Assay, Flow Cytometry, Imaging