Journal: Precision Clinical Medicine

Article Title: Agrimol B inhibits pancreatic ductal adenocarcinoma by induction of lethal mitophagy through decreasing mitochondrial transcription termination factor 3

doi: 10.1093/pcmedi/pbag009

Figure Lengend Snippet: Agrimol B blocks autophagic flux in PDAC cells. (A, B) Western blot analysis of P62 and CTSD in PANC-1 and AsPC-1 cells treated with Agrimol B for 24 h. (C, E, F) Immunofluorescence analysis of RFP-GFP-LC3 after PANC-1 and AsPC-1 cells were transfected with RFP-GFP-LC3 for 48 h, followed by treatment with or without Agrimol B for another 24 h. Scale bars, 10 μm. (D, G-L) Immunofluorescence analysis of the colocalization of endogenous LC3 with LAMP2 after treatment with Agrimol B or rapamycin for 24 h in PANC-1 and AsPC-1 cells. Scale bars, 10 μm. (M-O) Immunofluorescence analysis of LC3 in PDAC cells treated with or without Agrimol B in the presence or absence of HCQ. Scale bars, 10 μm.

Article Snippet: An anti-microtubule-associated protein light chain 3 (LC3) antibody (NB100-2220) was purchased from Novus, while anti-MTERF3 (EM1701-29) and lysosomal associated membrane protein 2 (LAMP2) (M1603-5) antibodies were purchased from HuaBio.

Techniques: Western Blot, Immunofluorescence, 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: 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 expression of lysosome 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 LAMP1 expression in murine mammary tissue. LAMP1-positive granule-like cells were observed under a light microscope, with brown staining in nuclei indicating positive signals. (B) Quantification of LAMP1-positive staining intensity in mammary tissues. (C) Using the same 6 experimental groups, immunohistochemical staining was performed to assess LAMP2 expression in mammary tissue. LAMP2-positive granule-like cells were observed under a light microscope, with brown nuclear staining indicating positive expression. (D) Quantification of LAMP2-positive staining intensity in mammary tissues. Scale bar = 10 μm. For (B, D) , 2-way ANOVA Dunnett’s multiple comparisons 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: Expressing, Infection, Control, Immunohistochemical staining, Staining, Light Microscopy

Journal: Frontiers in Neurology

Article Title: ATH-1105 mitigates multiple pathologies in ALS models both alone and in combination with riluzole

doi: 10.3389/fneur.2025.1582765

Figure Lengend Snippet: ATH-1105 enhances neuronal survival and neurite network integrity, and reduces cytoplasmic accumulation of extranuclear TDP-43, LC3 expression, and co-localization of TDP-43 and LC3 in glutamate-challenged motor neuron cultures. (A) Primary rat spinal motor neurons were treated with ATH-1105 and glutamate (5 μM) for 24 h and immunolabeled with microtubule-associated protein-2 (MAP2; neuronal marker), anti-TDP-43, and anti-LC3. Scale bar = 50 μm. Quantification of (B) motor neuron survival (number of MAP2 + neurons), (C) neurite network (total length of MAP2 + neurites), (D) cytoplasmic TDP-43 levels within MAP2 + staining, (E) LC3 + autophagosomes within MAP2 + staining, and (F) LC3/TDP-43 co-localization within MAP2 + staining, expressed as area per neuron. Data are expressed as percentage of normal control (100%) and presented as mean ± SEM; n = 4–6 (1 biological replicate). Statistical differences were determined by one-way ANOVA followed by Fisher’s LSD test versus glutamate control. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Fixed permeabilized cultures were incubated with the following primary antibodies: chicken anti MAP2 (1:400) (Abcam; #Ab5392), rabbit anti LC3b (1:50) (Abcam; #Ab192890), and mouse anti lysosomal-associated membrane protein 2 (LAMP2) (1:1000) (Novus; #NB300-591).

Techniques: Expressing, Immunolabeling, Marker, Staining, Control

Journal: Frontiers in Neurology

Article Title: ATH-1105 mitigates multiple pathologies in ALS models both alone and in combination with riluzole

doi: 10.3389/fneur.2025.1582765

Figure Lengend Snippet: ATH-1105 enhances neuronal survival and neurite network integrity, and reduces LAMP2 expression, LC3 expression, and co-localization of LAMP2 and LC3 in glutamate-challenged motor neuron cultures. (A) Primary rat spinal motor neurons were treated with ATH-1105 and glutamate (5 μM) for 24 h and immunolabeled with microtubule-associated protein-2 (MAP2; neuronal marker), anti-LAMP2, and anti-LC3. Scale bar = 50 μm. Quantification of (B) motor neuron survival (number of MAP2 + neurons), (C) neurite network (total length of MAP2 + neurites), (D) LAMP2 + lysosomes within MAP2 + staining, (E) LC3 + autophagosomes within MAP2 + staining, and (F) LC3/LAMP2 co-localization within MAP2 + staining, expressed as area per neuron. Data are expressed as percentage of normal control (100%) and presented as mean ± SEM; n = 4–6 (1 biological replicate). Statistical differences were determined by one-way ANOVA followed by Fisher’s LSD test versus glutamate control. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Fixed permeabilized cultures were incubated with the following primary antibodies: chicken anti MAP2 (1:400) (Abcam; #Ab5392), rabbit anti LC3b (1:50) (Abcam; #Ab192890), and mouse anti lysosomal-associated membrane protein 2 (LAMP2) (1:1000) (Novus; #NB300-591).

Techniques: Expressing, Immunolabeling, Marker, Staining, Control