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: 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