Journal: Nanomaterials

Article Title: Impact of Carbon Nano-Onions on Hydra vulgaris as a Model Organism for Nanoecotoxicology

doi: 10.3390/nano5031331

Figure Lengend Snippet: Raman spectra of p-CNO (black line), benz-CNO (red line), py-CNO (blue line) and py+-CNO (green line).

Article Snippet: Raman spectra were measured on a 800 UV LabRam Raman microscope (Horiba Jobin Yvon, Longjumeau, France).

Techniques:

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