Journal: bioRxiv

Article Title: Dysregulation of a novel autophagosome-mitochondria contact contributes to autophagy dysfunction and neurodegeneration in tauopathy

doi: 10.64898/2026.03.23.713823

Figure Lengend Snippet: Autophagosome/autophagic vacuole (AV)-mitochondria (Mito) contact hyper-tethering and AV retrograde transport defects in tauopathy axons. ( A and B ) Representative transmission electron microscopy (TEM) images ( A ) and quantitative analysis ( B ) of 8-month-old tauP301S Tg (PS19) hippocampi. The number of presynaptic AVs, the percentage of terminals containing AVs, and the number of AVs or mitochondria in AV-Mito contacts were quantified and normalized to or compared to those in non-Tg littermate controls. Data were quantified from a total number of presynaptic terminals ( n ) as indicated in parentheses ( B ) from three mice per group. Arrows: AV-Mito contacts; AV: autophagic vacuole; M: mitochondrion. ( C - F ) Representative time-lapse images and kymographs ( C ) and quantitative analysis ( D - F ) of non-Tg and tauP301S Tg axons after 24-hour trehalose (100 mM) incubation. AV-Mito contact duration and its frequency, the contact number per 100 μm axonal length, AV percentage in contacts, and the relative motility of AVs were quantified and compared between the two groups, respectively. Data were collected from three dissections: the total numbers of neurons ( n ) and AV-Mito contacts ( c ) are indicated in parentheses ( D - F ). Arrows: AVs (time-lapse images in top C ); AV-Mito contacts (kymographs in bottom C ). ( G ) Correlative light and electron microscope (CLEM) and cryo-electron tomography (cryo-ET) analysis of AV-Mito contacts in tauopathy axons. Low-magnification cryo-EM image of a tauP301S Tg neuron showing a region of the axonal process ( G1 ). CLEM of the region outlined by the white box in G1 , showing fluorescence images of mRFP-LC3-labeled AVs ( G2 ) and MitoView Green-marked mitochondria ( G3 ). The cryo-EM image of the same region acquired at 3,600 × magnification ( G4 ) is overlaid with the fluorescence image ( G5 ). The white dashed box indicates the potential AV-Mito contact site selected for tilt-series acquisition. Slice view of the reconstructed tomogram ( G6 ) with membrane annotations of a mitochondrion (green), an AV (red), synaptic vesicles (purple), and membranes (light grey) ( G7 ). Zoomed-in view of the AV-Mito contact site showed densities bridging the two membranes ( G8 , white arrows). Data were expressed as the mean ± SEM and analyzed using linear mixed-effects models. Scale bars: 100 nm ( A ) and 10 μm ( C ).

Article Snippet: Sources of antibodies or reagents are as follows: polyclonal antibodies against LC3 (Cell Signaling Technology, Cat# 2775), AMPKα (Cell Signaling Technology, Cat# 2532), p62/SQSTM1 (Abnova, Cat# H00008878-M01), NeuN (Millipore/Sigma, Cat# ABN78), TBC1D15 (Abcam, Cat# ab121396), Tau5 (DAKO, Cat# A0024), and synaptophysin/SYP (Abcam, Cat# ab32127); monoclonal antibodies against phospho-AMPKα (Cell Signaling Technology, Cat# 2535), AT8 (ThermoFisher Scientific, Cat# MN1020), syntaxin 1/STX1 (Santa Cruz Biotechnology, Cat# sc-12736), Rab7 (Sigma, Cat# R8779), GAPDH (Sigma, Cat# CB1001), TOM20 (Abcam, Cat# ab186734), mCherry (Takara, Cat# 632543), and Alexa fluor 488- (Cat# A-11017; Cat# A-11070), 546- (Cat# A-11018; Cat# A-11071), and 647- (Cat# A-21237; Cat# A-21246) conjugated secondary antibodies (Invitrogen); 10-NCP (VWR, Cat# 80017-188), trehalose (Cat# T0167), cycloheximide (Cat# 01810), rotenone (Cat# 557368), antimycin A (Cat# 1397-94-0), AICAR (Cat# A9978), CC (Cat# 171264), epoxomicin (Cat# 324800), CID 1067700 (Cat# SML0545), DMSO (Cat# D2650), and Sarkosyl (Cat# L9150) (Sigma); TBC1D15 shRNA (Cat# sc-154093-SH), Fis1 shRNA (Cat# sc-60644-SH), and control shRNA (Cat# sc-108060) (Santa Cruz Biotechnology); MitoView TM Green (Cat# 70054) (biotium); LentiBrite RFP-LC3 Lentiviral Biosensor (Cat# 17-10143) (Sigma).

Techniques: Transmission Assay, Electron Microscopy, Incubation, Microscopy, Tomography, Cryo-EM Sample Prep, Fluorescence, Labeling, Membrane

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Fenofibrate suppresses Mycoplasma bovis infection via autophagy-mediated cholesterol regulation in bovine mammary epithelial cells and murine mammary tissue

doi: 10.3389/fcimb.2025.1731492

Figure Lengend Snippet: Effects of doses and treatment durations of fenofibrate on autophagy by bMECs. (A) bMECs were treated with 50 μM fenofibrate for 0, 3, 6, 9, or 12 h, with HBSS serving as a positive control. Expression levels of lysosome-associated proteins LAMP2 and RAB7A, along with autophagy markers LC3 and SQSTM1, were assessed by Western blotting. Densitometric analysis was performed to quantify protein expression, normalized to GAPDH as a loading control. (B) bMECs were treated with 0, 10, 50, 100, or 200 μM fenofibrate for 9 h, with HBSS as a positive control. Protein levels of LAMP2, RAB7A, LC3, and SQSTM1 were evaluated by Western blotting. Relative expression was quantified by densitometry and normalized to GAPDH (for (A, B) , 1-way ANOVA Dunnett’s multiple comparisons tests and 2-tailed unpaired t -tests were used). Data are presented as mean ± SD from 3 independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Anti-LAMP1 antibody (67300-1-Ig), anti- lysosomal-associated membrane protein 2 (LAMP2) antibody (66301-1-Ig), anti-TFEB antibody (13372-1-AP), anti-TFE3 antibody (14480-1-AP), anti-LC3 polyclonal antibody (14600-1-AP), anti-ATG5 antibody (10181-2-AP), anti-RAB7A antibody (55469-1-AP), anti-mouse IgG-horseradish peroxidase (HRP) (SA00001-1), and Goat anti-rabbit IgG (SA00001-2) were all from Proteintech (Chicago, IL, USA).

Techniques: Positive Control, Expressing, Western Blot, Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Fenofibrate suppresses Mycoplasma bovis infection via autophagy-mediated cholesterol regulation in bovine mammary epithelial cells and murine mammary tissue

doi: 10.3389/fcimb.2025.1731492

Figure Lengend Snippet: Fenofibrate restores autophagic activity and autophagic flux in bMECs infected with M. bovis . (A) bMECs were divided into 6 experimental groups: control, M. bovis PG45 reference strain infection (MOI = 30, 9 hpi), M. bovis WT21 wild-type strain infection (MOI = 30, 9 hpi), fenofibrate treatment alone, fenofibrate combined with PG45 infection, and fenofibrate combined with WT21 infection. Expression levels of LAMP2, RAB7A, SQSTM1, and LC3, along with the loading control GAPDH, were evaluated by Western blot (using specific antibodies). (B) Densitometric analysis was performed to quantify relative protein levels of LAMP2, RAB7A, SQSTM1, and LC3-II, normalized to GAPDH. (C) Representative confocal images depict autophagic flux in mCherry-GFP-LC3-transfected bMECs across the 6 treatment groups: control, PG45-infected, WT21-infected, fenofibrate-treated, fenofibrate+PG45, and fenofibrate+WT21. Yellow puncta indicate autophagosomes, whereas red puncta represent autolysosomes. Nuclei were counterstained with Hoechst 33258 (blue). Scale bar = 10 μm. (D) Quantification of autophagosomes in bMECs. Twenty cells for each sample and at least 60 cells in each group were used for statistical analyses, Superscript ‘a’: Yellow puncta compared to the control group, Superscript ‘b’: Red puncta compared to the control group. For (A, B) , 2-way ANOVA Dunnett’s multiple comparisons tests were used; for (D) , 1-way ANOVA Dunnett’s multiple comparisons tests and 2-tailed unpaired t -tests were used. Data are presented as mean ± SD from 3 independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Anti-LAMP1 antibody (67300-1-Ig), anti- lysosomal-associated membrane protein 2 (LAMP2) antibody (66301-1-Ig), anti-TFEB antibody (13372-1-AP), anti-TFE3 antibody (14480-1-AP), anti-LC3 polyclonal antibody (14600-1-AP), anti-ATG5 antibody (10181-2-AP), anti-RAB7A antibody (55469-1-AP), anti-mouse IgG-horseradish peroxidase (HRP) (SA00001-1), and Goat anti-rabbit IgG (SA00001-2) were all from Proteintech (Chicago, IL, USA).

Techniques: Activity Assay, Infection, Control, Expressing, Western Blot, Transfection

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Fenofibrate suppresses Mycoplasma bovis infection via autophagy-mediated cholesterol regulation in bovine mammary epithelial cells and murine mammary tissue

doi: 10.3389/fcimb.2025.1731492

Figure Lengend Snippet: Fenofibrate affects co-localization of M. bovis , cholesterol, and LC3. (A) bMECs were divided into 6 groups: control, M. bovis PG45-infected, M. bovis WT21 wild-type strain-infected, fenofibrate-treated, fenofibrate + PG45-infected, and fenofibrate + WT21-infected. Representative confocal images illustrate triple staining with DiI to label M. bovis (red), GFP-LC3 to visualize the autophagy marker LC3 (green), and Filipin to detect cholesterol (blue). Scale bar = 10 μm.

Article Snippet: Anti-LAMP1 antibody (67300-1-Ig), anti- lysosomal-associated membrane protein 2 (LAMP2) antibody (66301-1-Ig), anti-TFEB antibody (13372-1-AP), anti-TFE3 antibody (14480-1-AP), anti-LC3 polyclonal antibody (14600-1-AP), anti-ATG5 antibody (10181-2-AP), anti-RAB7A antibody (55469-1-AP), anti-mouse IgG-horseradish peroxidase (HRP) (SA00001-1), and Goat anti-rabbit IgG (SA00001-2) were all from Proteintech (Chicago, IL, USA).

Techniques: Control, Infection, Staining, Marker

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Fenofibrate suppresses Mycoplasma bovis infection via autophagy-mediated cholesterol regulation in bovine mammary epithelial cells and murine mammary tissue

doi: 10.3389/fcimb.2025.1731492

Figure Lengend Snippet: Fenofibrate affects expression of autophagy markers in mammary tissue of mice infected with M. bovis . (A) Mice were allocated into 6 groups: control, M. bovis PG45-infected, M. bovis WT21 wild-type strain-infected, fenofibrate-treated, fenofibrate + PG45-infected, and fenofibrate + WT21-infected. Immunohistochemical staining was used to detect microtubule-associated protein 1 light chain 3 beta (LC3B) expression in murine mammary tissue. LC3B-positive granule-like cells were observed under a light microscope, with brown staining in nuclei indicating positive signals. (B) Quantification of LC3B-positive staining intensity in mammary tissues. (C) Using the same 6 experimental groups, immunohistochemical staining was performed to assess SQSTM1 expression in mammary tissue. SQSTM1-positive granule-like cells were observed under a light microscope, with brown nuclear staining indicating positive expression. (D) Quantification of SQSTM1-positive staining intensity in mouse mammary tissues. Scale bar = 50 μm. For (B, D) , 2-way ANOVA Dunnett’s multiple comparisons tests were used. Data are presented as mean ± SD from 3 independent experiments. NS (Not Significant) P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Anti-LAMP1 antibody (67300-1-Ig), anti- lysosomal-associated membrane protein 2 (LAMP2) antibody (66301-1-Ig), anti-TFEB antibody (13372-1-AP), anti-TFE3 antibody (14480-1-AP), anti-LC3 polyclonal antibody (14600-1-AP), anti-ATG5 antibody (10181-2-AP), anti-RAB7A antibody (55469-1-AP), anti-mouse IgG-horseradish peroxidase (HRP) (SA00001-1), and Goat anti-rabbit IgG (SA00001-2) were all from Proteintech (Chicago, IL, USA).

Techniques: Expressing, Infection, Control, Immunohistochemical staining, Staining, Light Microscopy

Journal: Revista da Associação Médica Brasileira

Article Title: Enoxaparin preserves cellular morphology and modulates mitophagy-associated Parkin–LC3 in acute ischemic rat myocardium

doi: 10.1590/1806-9282.20251136

Figure Lengend Snippet: Histological sections of Parkin and LC3 staining. a1—sham group, 40×, b1—control group, 40×, and c1—treatment group, 40×. Parkin staining: a2—sham group, 40×, b2—control group, 40×, and c2 treatment group—40×, LC3 staining. Parkin- and LC3-positive cells marked with black arrow.

Article Snippet: Nonspecific binding was prevented by incubation with a normal serum-blocking solution for 30 min. Next, sections were incubated overnight in a humidified chamber (30–60% humidity) with primary antibodies against LC3 (BiossUSA, LC3 Polyclonal Antibody, Cat. No: BS-4843R) and Parkin (BiossUSA, Parkin Polyclonal Antibody, Cat. No: BS-1865R).

Techniques: Staining, Control