Journal: Life Science Alliance

Article Title: Endolysosome-targeted nanoparticle delivery of antiviral therapy for coronavirus infections

doi: 10.26508/lsa.202403182

Figure Lengend Snippet: (A) General pipeline outlining FlowJo gating of viable, single cells using forward and side scattering, resulting in histogram frequency distribution of individual cell fluorescence. Positive cell gating is based on unstained and untreated controls; representative unstained and stained controls from the DQ-Red BSA assay are shown. (B, C) Rho-NP uptake measured by flow cytometry in Vero E6 cells and displayed as (B) representative intensity histogram or (C) median intensity bar graph. (D, E) Rho-NP uptake measured by flow cytometry in HFL1 cells and displayed as (D) representative intensity histogram or (E) median intensity bar graph. (F) Representative images of Vero E6 cells treated with NPs ± MFQ (100 μg/ml), free MFQ (20 μM), bafilomycin A1 (200 nM), or control and stained with Hoechst 33342 (blue) and LysoTracker Green (green) to probe for lysosome accumulation. Scale bars = 20 μm. (G) Quantification of lysosomal accumulation. Statistical significance was determined by one-way ANOVA: ** P < 0.01, *** P < 0.001, **** P < 0.0001 against untreated controls. All experiments represent N = 3 biological replicates, which represent the mean of at least n = 3 technical replicates. The intensity histogram displays a single representative cell population per timepoint. The intensity bar graph is displayed as medians ±SEM. All other experiments are displayed as means ± SEM. Source data are available for this figure.

Article Snippet: HFL1 cells were cultured in a 96-well plate at 15,000 cells/well for 24 h, after which the media were exchanged for media containing no-treatment control, empty NPs (12.5, 25, 50, 75, 100 μg/ml NPs), or MFQ-NPs (12.5, 25, 50, 75, 100 μg/ml NPs) for 24 h. Control treatments include bafilomycin A1 (200 nM), and Pepstatin A (10 μg/ml) + E64d (10 μg/ml) for 4 h, or free MFQ (10, 15 μM) for 24 h. After treatment, cells were washed with 1X PBS and incubated with 10 μg/ml DQ-Red BSA reagent (Thermo Fisher Scientific) in culture media for 1 h. After incubation with assay reagent, cells were trypsinized, washed with 1X PBS by centrifugation, resuspended in FACS buffer (PBS + 2% FBS), and then subjected to flow cytometry.

Techniques: Fluorescence, Staining, Flow Cytometry, Control

Journal: Life Science Alliance

Article Title: Endolysosome-targeted nanoparticle delivery of antiviral therapy for coronavirus infections

doi: 10.26508/lsa.202403182

Figure Lengend Snippet: (A) Representative confocal images of lysosomal pH measurements using LysoSensor Yellow/Blue dextran in Vero E6 cells treated with NP (±MFQ) 100 μg/ml, free MFQ 10 μM, bafilomycin 200 nM, or control. Scale bars = 10 μm. (B) Quantification of lysosomal pH. (C) Representative images of Calu-3 cells treated with NPs (±MFQ) 100 μg/ml, free MFQ 20 μM, bafilomycin 200 nM, or control and stained with Hoechst 33342 (blue) and LysoTracker Green (green) to probe for lysosome accumulation. Scale bars = 20 μm. (D) Quantification of lysosomal accumulation. (E) Quantification of lysosomal protease activity by DQ-Red BSA assay in Vero E6 cells treated with NPs (±MFQ), free MFQ, bafilomycin A1, pepstatin A + E64d, or controls at the indicated concentrations. Statistical significance was determined by one-way ANOVA: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 against untreated controls. Each independent LysoSensor imaging experiment represents the mean of n = 10–20 individual images. All other biological replicates represent the mean of n = 3 technical replicates. All experiments represent N = 3 biological replicates. All data are displayed as means ± SEM. Source data are available for this figure.

Article Snippet: HFL1 cells were cultured in a 96-well plate at 15,000 cells/well for 24 h, after which the media were exchanged for media containing no-treatment control, empty NPs (12.5, 25, 50, 75, 100 μg/ml NPs), or MFQ-NPs (12.5, 25, 50, 75, 100 μg/ml NPs) for 24 h. Control treatments include bafilomycin A1 (200 nM), and Pepstatin A (10 μg/ml) + E64d (10 μg/ml) for 4 h, or free MFQ (10, 15 μM) for 24 h. After treatment, cells were washed with 1X PBS and incubated with 10 μg/ml DQ-Red BSA reagent (Thermo Fisher Scientific) in culture media for 1 h. After incubation with assay reagent, cells were trypsinized, washed with 1X PBS by centrifugation, resuspended in FACS buffer (PBS + 2% FBS), and then subjected to flow cytometry.

Techniques: Control, Staining, Activity Assay, Imaging

Journal: International Journal of Oral Science

Article Title: Implantation awakens peri-implant osteogenic potential via Snx5-EGFR axis-mediated mechanical transduction

doi: 10.1038/s41368-025-00423-2

Figure Lengend Snippet: Inhibition of lysosomal degradation abolishes the enhanced osteogenic phenotype induced by Snx5 deficiency. a Micro-CT reconstructed images of bone architecture in Snx5 -KO mice under Sham and Imp conditions after Baf-A1 treatment ( n = 5). Yellow areas indicate ROIs, defined as annular zones extending approximately 75 μm to 175 μm outward from the implant surface; white solid lines delineate defect boundaries in the Sham group. Scale bar: 1 mm. b Quantitative analysis of BV/TV, BS/BV, and Tb.Th in Snx5 -KO mice under Sham and Imp conditions after Baf-A1 treatment ( n = 5). c Immunofluorescence staining and magnified views of peri-defect or peri-implant regions in Snx5 -KO mice after Baf-A1 treatment, showing Runx2 (green), LepR⁺ tdTomato⁺ (red), and DAPI (blue). Yellow dashed boxes indicate zoomed-in areas; white dashed lines outline defect or implant site boundaries ( n = 3). Scale bars: 100 μm. d Quantification of Runx2⁺ LepR⁺ tdTomato⁺ cells as a percentage of total LepR⁺ tdTomato⁺ cells ( n = 3). e Schematic illustration of the proposed mechanism: in Snx5 -KO cells, EGFR is primarily trafficked into Rab7⁺ late endosomes and degraded in LAMP1⁺ lysosomes. Treatment with the lysosomal inhibitor Baf-A1 blocks this pathway and attenuates the enhanced osteogenic phenotype. Baf-A1 Bafilomycin A1, Imp implant, ROI regions of interest. Data are presented as mean ± SD. ns not significant * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.000 1. Statistical analysis for panels b and d was performed using two-way ANOVA followed by Tukey’s multiple comparisons test

Article Snippet: In the Baf-A1 treatment group, 1 nmol/L Baf-A1 (HY-100558, MCE) was incorporated into the osteogenic induction medium, while an equivalent volume of DMSO (D103274, Aladin) was administered to the control cells.

Techniques: Inhibition, Micro-CT, Immunofluorescence, Staining