Journal: Innate immunity

Article Title: Src-family-tyrosine kinase Lyn is critical for TLR2-mediated NF-κB activation through the PI 3-kinase signaling pathway.

doi: 10.1177/1753425915586075

Figure Lengend Snippet: Figure 6. Lyn is essential to the activation of PI 3-kinase through phosphorylation of its p110 subunit. (A; Supplementary movies M1–M4) HEK 293–TLR2 cells were transfected with a fusion protein combining GFP and the PH domain of AKT. Transfected cells were either co-transfected with LynDK (500 ng/ml) or incubated with PP2. Cells were then stimulated with Pam3 (100 ng/ml) and the kinetics of the AKT–PH construct was observed by microvideoscopy using Zeiss Axiovert inverted microscope equipped with the Metafluor imaging system. Presented here are the images corresponding to 15 min of Pam3 stimulation. Controls correspond to cells incubated with DMSO and pcDNA empty vector, or cells incubated with LY294002 (25 mM), a specific inhibitor of PI 3-kinase. (B) HEK 293–TLR2 cells were transfected with pcDNA vector (vehicle) or LynDK (500 ng/ml) and stimulated with Pam3 (100 ng/ml). Lysates were immunoprecipitated with anti-Flag Abs and recruitment of PI 3-kinase to TLR2 was observed by Western blot with anti-p85a Abs. (C) Tyrosine phosphorylation of the p85a subunit was evaluated by Western blot in HEK 293–TLR2 transfected with LynDK (500 ng/ml) and stimulated with Pam3 (100 ng/ml). Anti-phosphotyrosine (4G10 clone) and anti-p85a Abs were used for immuno- precipitation and Western blot. (D) HEK 293–TLR2 were transfected with LynDK (500 ng/ml) and stimulated with Pam3 (100 ng/ml) and lysates were immunoprecipitated with either 4G10 or anti-p110 Abs. Phosphorylation of p110 was then revealed by Western blot with anti-p110 or 4G10 Abs. Controls correspond to cells transfected with pcDNA empty vector. (E) THP1–CD14 cells were incubated with PP2 (25mM) or DMSO, stimulated with Pam3 (100 ng/ml) and lysed. Phosphorylation of p110 catalytic subunit of PI 3-kinase was revealed by Western blot of THP1–CD14 lysates immunoprecipitated with either 4G10 or anti-p110 Abs. Controls correspond to cells treated with DMSO. These results are representative of three independent experiments.

Article Snippet: Polyclonal Abs to phospho-AKT (Ser473), AKT, phospho-P65 (Ser536), P65, phosphoP38, phospho-ERK, phospho-SAP-JNK, ERK, SAPJNK, IkB and mAb P38 were from Cell Signaling (Danvers, MA, USA). mAb to Flag was from Sigma. mAbs against CD14 and aminoacid 800-1139 of human p110 isoforms were from Santa Cruz Biotechnology.

Techniques: Activation Assay, Phospho-proteomics, Transfection, Incubation, Construct, Inverted Microscopy, Imaging, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: Innate immunity

Article Title: Src-family-tyrosine kinase Lyn is critical for TLR2-mediated NF-κB activation through the PI 3-kinase signaling pathway.

doi: 10.1177/1753425915586075

Figure Lengend Snippet: Figure 7. Lyn controls the PI 3-kinase pathway through phosphorylation of its p110 subunit after TLR2 engagement. The presence of bacteria-derived acylated lipoproteins results in the heterodimerization of TLR2 with TLR1/6 in membrane microdomains. MyD88/ IRAK are recruited to the receptor and lead to degradation of IkB and translocation of NF-kB subunits. A Rac/PI 3-kinase-dependent signaling pathway has also been described, including CD14 and Lyn that contribute to the activation cluster. After tyrosine phos- phorylation of the cytoplasmic domain of TLR2, the p85a subunit of PI 3-kinase is recruited to the receptor and phosphorylated on tyrosine. A Lyn-dependent tyrosine-phosphorylation is required to activate the PI 3-kinase catalytic subunit p110 that allows for recruitment of AKT to the inner membrane, precluding a cascade that results in transactivation of the p65 subunit of NF-kB. This leads to the nuclear translocation of a functional p50/p65 NF-kB heterodimer that results in the gene expression of pro-inflammatory cytokines.

Article Snippet: Polyclonal Abs to phospho-AKT (Ser473), AKT, phospho-P65 (Ser536), P65, phosphoP38, phospho-ERK, phospho-SAP-JNK, ERK, SAPJNK, IkB and mAb P38 were from Cell Signaling (Danvers, MA, USA). mAb to Flag was from Sigma. mAbs against CD14 and aminoacid 800-1139 of human p110 isoforms were from Santa Cruz Biotechnology.

Techniques: Phospho-proteomics, Bacteria, Derivative Assay, Membrane, Translocation Assay, Activation Assay, Functional Assay, Gene Expression

Journal: International Journal of Biological Sciences

Article Title: Polystyrene Nanoplastics Exacerbate HFD-induced MASLD by Reducing Cathepsin Activity and Triggering Large Vacuole Formation via Impaired Lysosomal Acidification

doi: 10.7150/ijbs.108268

Figure Lengend Snippet: Vacuole formation and lipid accumulation in PS-NP-treated liver cell lines. (A) Crystal violet staining was performed to confirm vacuole formation in response to 50 nm PS-NPs in indicated hepatocyte cell lines. Scale bar: 50 μm. (B) BODIPY staining in four cell lines (HepaRG, HepG2, SK-HEP-1, and 293T) treated with 100 μg/mL PS-NPs, demonstrating prominent lipid accumulation in HepaRG cells. Images were captured by a Leica microscope. Scale bar: 50 μm.

Article Snippet: Immunofluorescent images were captured using a confocal microscope (Zeiss, Germany; LSM 800).

Techniques: Staining, Microscopy

Journal: International Journal of Biological Sciences

Article Title: Polystyrene Nanoplastics Exacerbate HFD-induced MASLD by Reducing Cathepsin Activity and Triggering Large Vacuole Formation via Impaired Lysosomal Acidification

doi: 10.7150/ijbs.108268

Figure Lengend Snippet: Increased acidic compartments, lipid accumulation, and modulation of lipogenesis and lipolysis in PS-NP-treated HepaRG cells. (A) BODIPY and Nile Red staining were used to visualize neutral lipid accumulation in HepaRG cells treated with different PS-NP concentrations for 48 h. Scale bar: 50 μm. (B) Quantification of cellular fluorescence intensity for BODIPY and Nile Red staining. (C) Histogram overlay of flow cytometry BODIPY fluorescence measurements. (D) Intracellular triglyceride (TG) levels in HepaRG cells after 24 h of PS-NP exposure. Error bars represent the SEM. * p < 0.05 by ANOVA two-tailed unpaired t-test. (E) mRNA expression of hepatocyte nuclear factor 4 alpha ( HNF4A ), albumin ( ALB ), and cytochrome P450 3A4 ( CYP3A4 ) after 48 h of PS-NP incubation, normalized to RPL13A expression relative to 0 μg/mL PS-NPs. (F) Representative western blot images of CYP3A4 in HepaRG cells. (G) CYP3A4 activities in HepaRG cells exposed to varying PS-NP concentrations for 48 h. (H) Influence of 3-h pretreatment with 0.2 mM oleate on PS-NP-induced lipid accumulation. Scale bar: 200 μm. (I-J) Bar graphs showing the cellular fluorescence intensity of BODIPY (I) and LysoTracker (J) quantified using Gen5 software. Comparisons are made with the 0 μg/mL PS-NPs in with or without 0.2 mM oleate group. (K) Colocalization analysis of BODIPY with various organelle-specific markers (LysoTracker, MitoTracker, or ER-Tracker) in PS-NP-treated HepaRG cells. Images were acquired using a confocal microscope. Scale bar: 10 μm. (L-N) mRNA and protein levels of various genes associated with MASLD development. Error bars represent the SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 by one-way ANOVA and Tukey's multiple comparison tests. (N) Dose-dependent increase in lysosomal membraned protein LAMP1 level following PS-NP exposure.

Article Snippet: Immunofluorescent images were captured using a confocal microscope (Zeiss, Germany; LSM 800).

Techniques: Staining, Fluorescence, Flow Cytometry, Two Tailed Test, Expressing, Incubation, Western Blot, Software, Microscopy, Comparison

Journal: International Journal of Biological Sciences

Article Title: Polystyrene Nanoplastics Exacerbate HFD-induced MASLD by Reducing Cathepsin Activity and Triggering Large Vacuole Formation via Impaired Lysosomal Acidification

doi: 10.7150/ijbs.108268

Figure Lengend Snippet: Disturbance of autophagy flux induced by PS-NPs in MASLD development. (A) Increased S6 kinase 1 (S6K1) phosphorylation, an mTORC1 substrate. (B, C) mRNA (B) and protein (C) expression of p62, an autophagy substrate, and LC3B, a marker of autophagosomes. (D) Time-dependent protein expression of p62 and LC3B in PS-NP-treated HepaRG cells. Bar graphs show the intensity of each protein quantified using ImageJ software and normalized to β-actin; data are presented as the fold change relative to the 0 h sample. (E) Enhanced BODIPY-stained lipid accumulation induced by PS-NPs, accompanied by elevated immunofluorescence of p62 and LC3B. Scale bar: 50 μm. (F) Translocation to the nucleus of transcription factor EB (TFEB), an mTORC1 substrate. Scale bar: 10 μm. (G) Inhibition of PS-NP-induced vacuolization by rapamycin. HepaRG cells were pre-treated with 2 μg/mL rapamycin, followed by PS-NP exposure for 24 h, and then stained with crystal violet. Images were captured by a Leica microscope. Scale bar: 50 μm. (H) Changes in lipid accumulation (BODIPY) following treatment with autophagy regulators. HepaRG cells were pre-treated with 2 μg/mL rapamycin (an autophagy activator), followed by 10 nM Bafilomycin A1 (Baf A1; an autophagy inhibitor) for 3 h, and then PS-NPs. Scale bar: 50 μm. (I-J) Western blot and autophagy flux assessment. Effect of rapamycin (1 μg/mL) (I) and BafA1 (10 nM) (J) on PS-NP-triggered autophagy perturbations. Bar graphs depict the intensity of the p62 protein normalized to that of β-actin. (K) Examination of PS-NP-induced autophagy flux disturbances. (L) Accumulation of LC3B (a marker of autophagosomes, green) bound to the inner membrane of LAMP1-positive endolysosomes and autophagosomes in PS-NP-exposed HepaRG cells. Scale bar: 10 μm. Error bars represent the SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 by one-way ANOVA and Tukey's multiple comparison tests.

Article Snippet: Immunofluorescent images were captured using a confocal microscope (Zeiss, Germany; LSM 800).

Techniques: Phospho-proteomics, Expressing, Marker, Software, Staining, Immunofluorescence, Translocation Assay, Inhibition, Microscopy, Western Blot, Membrane, Comparison