Journal: Nature Communications

Article Title: Lysosome-targeting live attenuated influenza vaccines elicit robust and broad immunity in mice

doi: 10.1038/s41467-026-69920-0

Figure Lengend Snippet: a LTM-dependent degradation of viral NS1 protein. Western blot analysis shows reduced NS1 protein levels in cells expressing NS1-N LTM compared with the N LTM mutant , while NS1 mRNA levels remain comparable ( n = 3). b Lysosome dependence of LTM-mediated NS1 degradation. NS1-N LTM protein degradation is blocked by lysosomal inhibitors but not by proteasome or autophagy inhibition. HEK293T cells expressing either NS1-N LTM (left) or NS1-N LTM mutant (right) protein were cultured with or without the proteasome inhibitor MG-132 (10 µM), the autophagy inhibitor 3-methyladenine (3-MA; 10 mM), bafilomycin A1 (Baf A1; 0.4 µM), or chloroquine (CQ; 50 µM) for 6 h. The viral NS1 protein was detected by Western blotting ( n = 3). c Co-immunoprecipitation demonstrating interaction of HSC70 with NS1-N LTM but not with the NS1 LTM mutant ( n = 3). d Dependence of LTM-mediated NS1 degradation on LAMP2A. Conventional HEK293T cells and LAMP2A-KO HEK293T cells were transfected with constructs expressing NS1-N LTM (left) or NS1-N LTM mutant (right) protein and collected 24 h post-transfection. Viral NS1 protein was detected by Western blotting ( n = 3). Immunofluorescence analysis showing colocalization of NS1-N LTM with HSC70 ( e ) and LAMP2A ( f ), but not of the NS1-N LTM mutant . Green, NS1; red, HSC70 or LAMP2A; blue, nuclei; yellow, colocalization sites; scale bar, 5 µm. Representative images of at least three independent experiments are shown. LAMP2A-dependent degradation of NS1-N LTM during viral infection in conventional and LAMP2A-KO HEK293T ( g ) and A549 ( h ) cells. Replication competence of NS1-N LTM and NS1-N LTM mutant viruses in conventional and LAMP2A-KO HEK293T ( i ) and A549 ( j ) cells. Immunofluorescence staining of influenza viral M1 protein at 48 h after infection (MOI = 0.01) showing the replication competence of NS1-N LTM or NS1-N LTM mutant virus in conventional and LAMP2A-KO cells. Green, M1; blue, nuclei; scale bar, 100 µm. Viral titers in culture supernatants were quantified by immunofluorescence focus-forming unit (FFU) assay ( n = 3). Data are means ± s.d; n = 3 biologically independent experiments; unpaired two-tailed t -test for ( i ) and ( j ); *** P < 0.001. Source data are provided as a Source Data file.

Article Snippet: After another 6 h of culture, the cells were washed with PBS, fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature, washed with PBS, permeabilized with PBS containing 0.1% Triton X-100 for 5 min, blocked with PBS containing with 0.1% Triton X-100 and 10% goat serum for 1 h at room temperature, and incubated with anti-Flag antibody (MBL, Cat# PM020; 1:1000 dilution) and anti-HSC70 antibody (Santa Cruz Biotechnology, Cat# sc-7298; 1:300 dilution) or anti-LAMP2A antibody (Santa Cruz Biotechnology, Cat# sc-18822; 1:300 dilution) diluted in PBS containing 0.1% Triton X-100 and 1% goat serum overnight at 4 °C, followed by incubation with Alexa Fluor 488 goat anti-rabbit IgG (Life Technologies, Cat# A11034; 1:1000 dilution) and Alexa Fluor 555 goat anti-mouse IgG (Life Technologies, Cat# A32727; 1:1000 dilution) for 1 h at room temperature.

Techniques: Western Blot, Expressing, Mutagenesis, Inhibition, Cell Culture, Immunoprecipitation, Transfection, Construct, Immunofluorescence, Infection, Staining, Virus, Two Tailed Test

Journal: Nature Communications

Article Title: Lysosome-targeting live attenuated influenza vaccines elicit robust and broad immunity in mice

doi: 10.1038/s41467-026-69920-0

Figure Lengend Snippet: a Schematic illustration depicting the design and generation of LYTAR 2.0 influenza viruses. LYTAR 2.0 viruses are attenuated in conventional cells through LTM-mediated viral protein degradation by the CMA system but can replicate efficiently in LAMP2A-knockout (KO) cells. VP stands for viral protein. b Western blots showing the dependence of LTM-mediated viral protein degradation on the lysosome. HEK293T cells expressing the indicated viral proteins were cultured in the presence or absence of Baf A1 (0.4 µM) for 6 h. The viral proteins were detected by Western blotting ( n = 3). c Western blots showing the LAMP2A dependency of LTM-mediated viral protein degradation. Conventional HEK293T cells and LAMP2A-KO HEK293T cells were transfected with constructs expressing the indicated viral proteins and collected 24 h post-transfection for viral protein detection by Western blotting ( n = 3). d Western blots showing the LAMP2A dependency of LTM-mediated viral protein degradation in HEK293T cells. Conventional HEK293T cells and LAMP2A-KO HEK293T cells were infected with the indicated LYTAR 2.0 viruses and collected 48 h post-infection for detection of viral proteins by Western blotting ( n = 3). e Western blots showing the LAMP2A dependence of LTM-mediated viral protein degradation in A549 cells. Conventional A549 cells and LAMP2A-KO A549 cells were infected with the indicated LYTAR 2.0 viruses and collected 48 h post-infection for detection of viral proteins by Western blotting ( n = 3). Source data are provided as a Source Data file.

Article Snippet: After another 6 h of culture, the cells were washed with PBS, fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature, washed with PBS, permeabilized with PBS containing 0.1% Triton X-100 for 5 min, blocked with PBS containing with 0.1% Triton X-100 and 10% goat serum for 1 h at room temperature, and incubated with anti-Flag antibody (MBL, Cat# PM020; 1:1000 dilution) and anti-HSC70 antibody (Santa Cruz Biotechnology, Cat# sc-7298; 1:300 dilution) or anti-LAMP2A antibody (Santa Cruz Biotechnology, Cat# sc-18822; 1:300 dilution) diluted in PBS containing 0.1% Triton X-100 and 1% goat serum overnight at 4 °C, followed by incubation with Alexa Fluor 488 goat anti-rabbit IgG (Life Technologies, Cat# A11034; 1:1000 dilution) and Alexa Fluor 555 goat anti-mouse IgG (Life Technologies, Cat# A32727; 1:1000 dilution) for 1 h at room temperature.

Techniques: Knock-Out, Western Blot, Expressing, Cell Culture, Transfection, Construct, Infection

Journal: Nature Communications

Article Title: Lysosome-targeting live attenuated influenza vaccines elicit robust and broad immunity in mice

doi: 10.1038/s41467-026-69920-0

Figure Lengend Snippet: a Multi-cycle replication kinetics of the indicated viruses in conventional and LAMP2A-KO MDCK cells. Data are presented as means ± s.d ( n = 3). b triLTM-dependent degradation of viral proteins. Western blot analysis shows reduced levels of triLTM-tagged viral proteins compared with mutated triLTM-tagged controls, while corresponding mRNA levels remain comparable ( n = 3). c Lysosome dependence of triLTM-mediated viral protein degradation. HEK293T cells expressing triLTM-tagged or mutated triLTM-tagged viral proteins were cultured in the presence or absence of Baf A1 (0.4 µM) for 6 h and collected for detection of indicated viral proteins by Western blotting ( n = 3). d Co-immunoprecipitation demonstrating interaction of HSC70 with triLTM-tagged viral proteins but not with mutated triLTM-tagged viral proteins ( n = 3). LAMP2A-dependent degradation of triLTM-tagged viral proteins during viral infection in conventional and LAMP2A-KO HEK293T ( e ) and A549 ( f ) cells ( n = 3). Conventional cells and LAMP2A-KO cells were infected with LYTAR 2.0 dual triLTMs or LYTAR 2.0 dual triLTMs mutant virus and collected at 48 h after infection for detection of indicated proteins by Western blotting ( n = 3). Replication competence of LYTAR 2.0 dual triLTMs and LYTAR 2.0 dual triLTMs mutant viruses in conventional and LAMP2A-KO HEK293T ( g ) and A549 ( h ) cells. Immunofluorescence staining of influenza viral M1 protein at 48 h after infection (MOI = 0.01) showing the replication competence of LYTAR 2.0 dual triLTMs and LYTAR 2.0 dual triLTMs mutant virus in conventional and LAMP2A-KO HEK293T cells. Green, M1; blue, nuclei; scale bar, 100 µm. Viral titers in culture supernatants were quantified by immunofluorescence focus-forming unit (FFU) assay ( n = 3). Data are means ± s.d; n = 3 biologically independent experiments; unpaired two-tailed t -test for ( g ) and ( h ); *** P < 0.001. Source data are provided as a Source Data file.

Article Snippet: After another 6 h of culture, the cells were washed with PBS, fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature, washed with PBS, permeabilized with PBS containing 0.1% Triton X-100 for 5 min, blocked with PBS containing with 0.1% Triton X-100 and 10% goat serum for 1 h at room temperature, and incubated with anti-Flag antibody (MBL, Cat# PM020; 1:1000 dilution) and anti-HSC70 antibody (Santa Cruz Biotechnology, Cat# sc-7298; 1:300 dilution) or anti-LAMP2A antibody (Santa Cruz Biotechnology, Cat# sc-18822; 1:300 dilution) diluted in PBS containing 0.1% Triton X-100 and 1% goat serum overnight at 4 °C, followed by incubation with Alexa Fluor 488 goat anti-rabbit IgG (Life Technologies, Cat# A11034; 1:1000 dilution) and Alexa Fluor 555 goat anti-mouse IgG (Life Technologies, Cat# A32727; 1:1000 dilution) for 1 h at room temperature.

Techniques: Western Blot, Expressing, Cell Culture, Immunoprecipitation, Infection, Mutagenesis, Virus, Immunofluorescence, Staining, Two Tailed Test

Journal: American Journal of Physiology - Cell Physiology

Article Title: Intestinal epithelial tight junction barrier regulation by autophagy-related protein ATG6/beclin 1

doi: 10.1152/ajpcell.00246.2018

Figure Lengend Snippet: Tat-beclin 1 increases the rate of occludin endocytosis. A: following surface biotinylation assay, as detailed in experimental procedures, and SDS-PAGE, immunoblots were probed with anti-occludin antibody. Caco-2 cells treated with Tat-beclin 1 peptide showed an increased amount of biotinylated occludin. B: %endocytosed occludin compared with total occludin contents at indicated time points, from 3 independent experiments. *P < 0.01 vs. Tat-scrambled peptide, unpaired Student’s t-test; n = 4, representation of 3 independent experiments. C: Tat-beclin 1 induces increased lysosomal targeting of occludin. In Tat-beclin 1-treated cells, occludin showed increased colocalization with lysosomal marker lysosome-associated membrane protein-2 (LAMP2) compared with Tat-scrambled peptide (Tat-scr)-treated cells. Occludin, green; LAMP2, red. Only merged signals (yellow) are retained in the colocalization images. Scale bars = 5 μm. Representation of 20 areas examined in 3 independent experiments.

Article Snippet: Primary antibodies used in this study included occludin (33-1500; Invitrogen; and LS-C-140253-100; LifeSpan BioSciences), zona occludens protein-1 (ZO-1, 617300; Invitrogen), claudin-1 (51-9000; Invitrogen), claudin-2 (51-6100; Invitrogen), claudin-3 (34-1700; Thermo Fisher Scientific), claudin-4 (PA-5-16875; Thermo Fisher Scientific), caveolin-1 (3238; Cell Signaling Technology), early endosome antigen 1 (EEA1, E-7659; Sigma-Aldrich), Ras-related protein Rab-5 (Rab5, R-4654; Sigma-Aldrich), beclin 1 (ab-114071 and ab-207612; Abcam), microtubule-associated protein 1A/1B-light chain 3B (LC3B, L-7543; Sigma-Aldrich), ATG16 (PA5-35207; Thermo Fisher Scientific), lysosome-associated membrane protein-2 (LAMP2, NBP-2-22217; Novus Biologicals), and β-actin (MA-5-15739; Thermo Fisher Scientific).

Techniques: Surface Biotinylation Assay, SDS Page, Western Blot, Marker, Membrane

Journal: Autophagy

Article Title: Autophagy is essential for ultrafine particle-induced inflammation and mucus hyperproduction in airway epithelium

doi: 10.1080/15548627.2015.1124224

Figure Lengend Snippet: Autophagic flux differentially modulates PM-induced expression of IL8 and MUC5AC in HBE cells. (A to C) Cells were treated with PM (100 μg/ml) with or without Baf A1 (10 nM) for 24 h, and were analyzed for the atuophagic vesicles by TEM (A), or were measured for the mRNA levels of IL8 (B) or MUC5AC (C). (D and E) Cells were transfected with Control- or LAMP2-siRNA for 24 h, and then were treated with PM (100 μg/ml) for an additional 24 h to examine the mRNA levels of IL8 (D) or MUC5AC (E). Data are presented as Mean ± SEM of 3 independent experiments. *, P < 0.05.

Article Snippet: The initial set of siRNAs of Control(sc-37007), ATG5 (sc-41445) , BECN1 (sc-29797) , RELA (sc-29410) , LAMP2 (sc-29390) , JUN (sc-29223) were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Transfection, Control

Journal: Autophagy

Article Title: Autophagy is essential for ultrafine particle-induced inflammation and mucus hyperproduction in airway epithelium

doi: 10.1080/15548627.2015.1124224

Figure Lengend Snippet: The NFKB1 and AP-1 pathways differentially mediate PM-induced expression of IL8 and MUC5AC. Cells were treated with CHUK inhibitor VII (A and B) or AP-1 inhibitor SP600125 (E and F) together with PM (100μg/ml) for 24 h, or treated with RELA- (C and D) or JUN-siRNA (G and H) for 24 h, and then with PM (100 μg/ml) for an additional 24 h. The mRNA expression of IL8 (A, C, E, and G) and MUC5AC (B, D, F, and H) were measured. Data are presented as Mean ± SEM. *, P < 0.05; ***, P < 0.001; n.s., not significant. (E and F) Cells were treated with indicated Control- or LAMP2-siRNA for 48 h, and then with PM (100 μg/ml) for an additional 12 h. The protein levels of RELA, NFKBIA (E) or p-JUN (F) were examined by western blot. Images are representative of 3 independent experiments.

Article Snippet: The initial set of siRNAs of Control(sc-37007), ATG5 (sc-41445) , BECN1 (sc-29797) , RELA (sc-29410) , LAMP2 (sc-29390) , JUN (sc-29223) were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Control, Western Blot

Journal: Scientific Reports

Article Title: Loss of chaperone-mediated autophagy is associated with low vertebral cancellous bone mass

doi: 10.1038/s41598-022-07157-9

Figure Lengend Snippet: Macroautophagy and proteasomal degradation increase in the absence of CMA. Calvarial osteoblasts were isolated from Lamp2AC global knock-out (L2ACgKO) mice and their control littermates (wild-type mice, WT). ( a ) Calvarial osteoblasts isolated from one mouse per genotype were plated in 6 well plates and cultured with vehicle (DMSO) or 100 μM Bafilomycin A (BafA) for 4 h. LAMP2A, LC3, and p62 protein levels were determined by western blot analysis. ( b ) LC3-II and p62 levels were quantified and normalized to actin levels. For LC3-II flux analysis BafA treatment of each genotype was normalized its vehicle treatment. n = 3 wells/treatment per genotype. Lines indicate mean ± STDEV. For comparisons of LC3-II/actin and p62/bactin levels p values were calculated and evaluated by Two-way ANOVA. For LC3-II flux comparison p value was calculated and evaluated by student’s t -test; *, p < 0.05. This experiment was repeated using calvarial cells isolated from another set of WT and L2ACgKO mice. These results can be found in S. Fig. c. (c,d ) Calvarial osteoblasts isolated from one mouse per genotype were plated in 6 well plates and cultured with vehicle (DMSO) or 50 μM of proteasome inhibitor MG132 for 6 h. The levels of ubiquitinated proteins and K48-linked ubiquitinated proteins were quantified by Western blot analysis. Full-length blots can be found in S. Fig. .

Article Snippet: The primary antibodies used were LAMP2A (Novus Biologicals, # NBP267298), LC3 (Cell Signaling Technology, # 12741), β-Actin (Millipore Sigma, A5316), Ubiquitin (Cell Signaling Technology, #43124), K48-Ubiquitin (Cell Signaling Technology, #8081).

Techniques: Isolation, Knock-Out, Control, Cell Culture, Western Blot, Comparison

Journal: Scientific Reports

Article Title: Loss of chaperone-mediated autophagy is associated with low vertebral cancellous bone mass

doi: 10.1038/s41598-022-07157-9

Figure Lengend Snippet: Differential expression of Lamp2 isoforms in different tissues.

Article Snippet: The primary antibodies used were LAMP2A (Novus Biologicals, # NBP267298), LC3 (Cell Signaling Technology, # 12741), β-Actin (Millipore Sigma, A5316), Ubiquitin (Cell Signaling Technology, #43124), K48-Ubiquitin (Cell Signaling Technology, #8081).

Techniques: Quantitative Proteomics

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 Immunology

Article Title: Hyperammonemia increases the release of pathological extracellular vesicles from monocytes by impairing lysosomal function and autophagy through the TNFα–cAMP–PKA–LC3 pathway

doi: 10.3389/fimmu.2025.1724800

Figure Lengend Snippet: Monocytes from hyperammonemic rats exhibit lysosomal dysfunction-related markers, which are regulated by PKA and TNFα pathways. (A) Representative confocal microscopy images of LysoTracker Red dye (red) in monocyte cells (bright field). (B) Quantification of the mean fluorescence intensity of LysoTracker Red dye in monocyte cells (n= 6). Protein content of (C) Cathepsin-L (n=), (D) Lamp2 (n= 6-11), and (E) LC3 (n= 3-8) in monocyte cells, assessed by Western blot, expressed as percentage of the control group. Representative images of the blots of each protein are shown. All data are presented as mean ± SEM. Statistical comparisons between C-M-EV and HA-M-EV groups were performed using an unpaired t-test. Statistical analysis was determined using four separate one-way ANOVAs, each comparing the two baseline groups (C, HA) with one of the treatment groups (C-M-EV + Forsk, HA-M-EV + PKAi, C-M-EV + TNFα, or HA-M-EV + aTNFα). One-way ANOVA followed by Fisher’s (for Cathepsin-L, Lamp2, LC3) or by Tukey’s (for lysotracker) post-hoc tests were performed to compare all groups. Values significantly different from the C group are indicated by asterisk (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001), and values significantly different from HA group are indicated by a (a=p<0.05, aa=p<0.01, aaaa=p<0.0001). Scale bar = 5 µm, as indicated in the figure.

Article Snippet: Primary antibodies used were against Alix (1:500, ProteinTech, #12422-1-AP), BDNF (brain-derived neurotrophic factor, 1:1,000, Invitrogen, #OSB00017W), Cathepsin-L (1:200, R&D #AF1515), CCL2 (1:2,000, Proteintech, #66272-1), Flotillin-2 (1:500, Invitrogen, #PA5-17178), IL-1β (1:500, RD Systems, #AF-501-NA), LAMP2 (1:200, Novus Biologicals, #NB300-591), LC3 (microtubule-associated protein 1A/1B-light chain 3, 1:1,000, Novus Biologicals, NB100-2220), S1PR2 (Sphingosine-1-Phosphate Receptor Type 2, 1:1,000, Proteintech, #21180-1-AP), TNFα (1:500, RD Systems, #AF-510-NA), TNFR1 (1:1,000, Abcam, #ab19139), TrkB (tyrosine receptor kinase B, 1:500, Abcam, #ab18987), and β-actin (1:5000, Abcam, #ab6276) or GAPDH (glyceraldehyde-3-phosphate dehydrogenase, 1:5,000, Millipore, #MAB374) as loading control.

Techniques: Confocal Microscopy, Fluorescence, Western Blot, Control

Journal: Frontiers in Immunology

Article Title: Hyperammonemia increases the release of pathological extracellular vesicles from monocytes by impairing lysosomal function and autophagy through the TNFα–cAMP–PKA–LC3 pathway

doi: 10.3389/fimmu.2025.1724800

Figure Lengend Snippet: Schematic representation of the proposed mechanism by which (1) hyperammonemia induces the formation of pathological EV containing TNFR1-TNFα in monocytes, (2) how TNFα and PKA modulate this process, and (3) how these EV induce pathological effects in the hippocampus. Hyperammonemia increases TNF α and activation of its receptor TNFR1 in monocytes, leading to activation of adenylate cyclase and increased cAMP levels and PKA activation. PKA alters both LC3, leading to increased lysosomal pH and impaired expression of lysosomal proteins such as cathepsin-L and LAMP2. This results in lysosomal dysfunction and altered autophagic flux enhances the release from multivesicular bodies (MVBs) of EV containing increased levels of TNFR1 and TNFα. These pathological HA-M-EV enhance activation of the TNFα–TNFR1–S1PR2–IL-1β–BDNF pathway and the TrkB–NR2B–GluA1–GluA2 pathway in hippocampal slices from control rats, inducing neuroinflammation and altered glutamatergic neurotransmission. All these effects are reversed by blocking TNFα with anti-TNFα or inhibiting PKA with H69 in the cultures of monocytes from hyperammonemic rats (green symbols). Activation of TNFR1 with recombinant TNFα (rTNFα) or of adenylate cyclase with forskolin in monocyte cultures from control rats induce essentially the same effects (red arrows). This figure was created using Biorender.com .

Article Snippet: Primary antibodies used were against Alix (1:500, ProteinTech, #12422-1-AP), BDNF (brain-derived neurotrophic factor, 1:1,000, Invitrogen, #OSB00017W), Cathepsin-L (1:200, R&D #AF1515), CCL2 (1:2,000, Proteintech, #66272-1), Flotillin-2 (1:500, Invitrogen, #PA5-17178), IL-1β (1:500, RD Systems, #AF-501-NA), LAMP2 (1:200, Novus Biologicals, #NB300-591), LC3 (microtubule-associated protein 1A/1B-light chain 3, 1:1,000, Novus Biologicals, NB100-2220), S1PR2 (Sphingosine-1-Phosphate Receptor Type 2, 1:1,000, Proteintech, #21180-1-AP), TNFα (1:500, RD Systems, #AF-510-NA), TNFR1 (1:1,000, Abcam, #ab19139), TrkB (tyrosine receptor kinase B, 1:500, Abcam, #ab18987), and β-actin (1:5000, Abcam, #ab6276) or GAPDH (glyceraldehyde-3-phosphate dehydrogenase, 1:5,000, Millipore, #MAB374) as loading control.

Techniques: Activation Assay, Expressing, Control, Blocking Assay, Recombinant