Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Interferon Alpha Induces Cellular Autophagy and Modulates Hepatitis B Virus Replication

doi: 10.3389/fcimb.2022.804011

Figure Lengend Snippet: IFNα-2a induces autophagy through inhibiting Akt/mTOR pathway and blocks autophagic degradation. (A) HepG2.2.15 cells were treated with 6000 U/ml IFNα-2a. After 48 hr, cells were fixed, incubated with rabbit anti-LC3 antibodies, followed by staining with Alexa Fluor 488-conjugated anti-rabbit secondary antibody IgG. Finally, the distribution of LC3 was imaged by confocal microscopy. Cells treated with 10 nM chloroquine (CQ) were used as a positive control. LC3 puncta in cells were quantified as described previously ( Lin et al., 2020 ). Scale bar: 5 μm. (B) HepG2.2.15 cells were treated with the indicated concentrations of IFNα-2a and harvested after 48 hr. HepG2.2.15 cells were cultured in the medium either with 6000 U/ml IFNα-2a or rapamycin (1 μM) for 48 hr. p62 and LC3 were analyzed by western blotting using beta‐actin as a loading control. The relative levels were determined by quantifying the gray scales of bands, using beta‐actin as a loading control. (C) HepG2.2.15 cells were treated with 6000 U/ml IFNα-2a for 48 hr, followed by incubation with 10 μg/mL DQ-BSA for 30 minutes. The fluorescent signal of DQ-BSA was detected by confocal microscopy. Cells cultured with EBSS for 2 hours were used as a positive control and CQ treatment was as a negative control. Scale bar: 5 μm. (D) PHHs were infected with HBV virions (multiplicity of infection = 30). 4 days post infection, PHHs were treated with IFNα-2a twice (1000 U/ml and 6000 U/ml). After 48 hr, p62 and LC3 were analyzed by western blotting using beta‐actin as a loading control. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: The fluorescent signal produced by lysosomal proteolysis of DQ Red BSA was quantified with an LSM 710 confocal microscope (Zeiss, Germany).

Techniques: Incubation, Staining, Confocal Microscopy, Positive Control, Cell Culture, Western Blot, Negative Control, Infection

Journal: Molecular Cancer

Article Title: Cathepsin S-mediated autophagic flux in tumor-associated macrophages accelerate tumor development by promoting M2 polarization

doi: 10.1186/1476-4598-13-43

Figure Lengend Snippet: Cat S inhibition causes defective autophagy flux in macrophage in the tumor microenvironment. (A) WT or Cat S -/- macrophages were transfected with Cherry-GFP-LC3, then cocultured with SL4 cells for 48 hrs and serum-starved for at least 12 hrs before co-culture. Confocal fluorescence was used to image autophagosomes (red and green foci) and autolysosomes (red-only foci). Scale bars = 10 μm. Three independent experiments were performed. (B) WT or Cat S -/- macrophages expressing the GFP-LC3 reporter construct were seeded in plastic wells, then recognition and counting by fluorescence image analysis using the KineticScan HCS Reader. Data are mean ± SEM of 3 independent experiments. **, P <0.01. NS indicates not significant. (C) WT or Cat S -/- macrophages were cocultured with SL4 cells and serum-starved for at least 12 hrs before co-culture, and WT macrophages in the presence or absence of Cat S inhibitor Z-FL-COCHO (10 μmol/L). WT or Cat S -/- macrophages were loaded with 10 μg/mL DQ-Green BSA for 15 min, washed twice and incubated in media for the indicated times, then subjected to flow cytometry analysis. Background (gray peak) represents samples without the DQ-Green BSA loading. Three independent experiments were performed. (D) WT or Cat S -/- macrophages were placed on coverslips as above in C, and incubated in media containing DQ-Green BSA (10 μg/mL) for 15 min. Cells were washed twice with PBS and incubated in media for the indicated times. Cells were fixed and the fluorescent degradation products of the DQ-Green BSA in lysosome were imaged using confocol images analysis. Scale bars = 5 μm. Dotted lines indicate the cell margin. Three independent experiments were performed.

Article Snippet: Cells were harvested, and Green-fluorescent of DQ-BSA was analyzed by flow cytometry using a FACSCalibur flow cytometer (BD Biosciences) and CellQuest (BD Biosciences) and FlowJo (Treestar) software.

Techniques: Inhibition, Transfection, Co-Culture Assay, Fluorescence, Expressing, Construct, Incubation, Flow Cytometry

Journal: bioRxiv

Article Title: A mycobacterial Sec61 inhibitor disrupts lysosome function by blocking Vacuolar-ATPase biosynthesis

doi: 10.1101/2025.08.26.671788

Figure Lengend Snippet: A. Schematic of LC3 tandem tag assay. B-D. HeLa cells were incubated with 0.05% DMSO for 32hr or 31.25ng/ml mycolactone (Myco) for various times as indicated with or without 300nM Bafilomycin A1 (BAFA1) for 4hr. B. Cells transiently transfected with pDEST-mcherry-eGFP-LC3B before treatment with mycolactone as described followed by live cell imaging, with the mCherry fluorescence pseudocoloured as magenta and eGFP as green. Quantitation shows the total number per cell (left hand graph, data representative of n=3 independent experiments; ordinary 2-way ANOVA) and relative proportions of magenta and white puncta (right hand graph, mean of n=3 biological replicates ± SEM; RM 2-way ANOVA) visualised in cells expressing mCherry-eGFP-LC3B. Scale bar =20µm. Data are compared to DMSO in the absence and presence of BAFA1 separately, and the effect of BAFA1 on each treatment is compared (indicated in blue). C. Cells were incubated with 20µg/ml DQ Red BSA for 4hr before washing and imaging for the unquenched signal. Scale bar =50µm. Quantitation of mean fluorescence per cell for at least 178 cells (data representative of n=3 independent experiments, ordinary 1-way ANOVA). D. Cells were fixed with 4% PFA, permeabilised, stained with anti-TFEB antibodies (grey) and counterstained with DAPI. Scale bar = 20µm. Graph shows mean % cells with nuclear TFEB staining from n=3 biological replicates ± SEM. ns; not significant, *; p<0.05, ***; p<0.001, ****; p<0.0001.

Article Snippet: DQ Red BSA (20μg/ml) (Fisher Scientific) was added in Optimem (Fisher Scientific) 4hr before the end of the experiment.

Techniques: Incubation, Transfection, Live Cell Imaging, Fluorescence, Quantitation Assay, Expressing, Imaging, Staining

Journal: bioRxiv

Article Title: A mycobacterial Sec61 inhibitor disrupts lysosome function by blocking Vacuolar-ATPase biosynthesis

doi: 10.1101/2025.08.26.671788

Figure Lengend Snippet: A. Cells were transiently transfected with pDEST-mcherry-eGFP-LC3B before treatment with mycolactone as described in Fig1A. Quantitation shows the total number of magenta and white puncta per cell. Data represents the mean of 3 independent experiments ± SEM analysed by RM 2-way ANOVA. Data are compared to DMSO in the absence and presence of BAFA1 separately, and the effect of BAFA1 on each treatment is compared (in blue). B. HeLa cells were incubated with 0.05% DMSO or 31.25ng/ml mycolactone (Myco) for various times, stained with CellEvent and propidium iodide and imaged. Data represents the % viable cells at each time point and is the mean of n=3 independent experiments ± SEM (RM one-way ANOVA). C. BMDM were seeded onto IBIDI 8-well chamber-slides and incubated with 0.05% DMSO for 24hr, 31.25ng/ml mycolactone (Myco) for various times or 200nM Bafilomycin A1 (BAFA1) for 4hr. DS Red BSA was added for 4hr before washing and imaging. Scale bar =50µm. Data represents median DS Red BSA fluorescence per cell for at least 48 cells ± IQR. Data representative of n=3 independent experiments (Kruskal-Wallis test). D. THP-1 cells were plated onto IBIDI 8-well chamber slides in the presence of 20ng/ml PMA. After 72hr medium was replaced and cells incubated for 24hr before adding 0.05% DMSO for 24hr, 31.25ng/ml mycolactone (Myco) for various times or 200nM Bafilomycin A1 (BAFA1) for 4hr. DS Red BSA was added for 4hr before washing and imaging. Scale bar =50µm. Data represents median DS Red BSA fluorescence per cell for at least 53 cells ± IQR. Data representative of n=3 independent experiments (Kruskal-Wallis test). ns; not significant, *; p<0.05, **; p<0.01, ***; p<0.001, ****; p<0.0001 or as indicated.

Article Snippet: DQ Red BSA (20μg/ml) (Fisher Scientific) was added in Optimem (Fisher Scientific) 4hr before the end of the experiment.

Techniques: Transfection, Quantitation Assay, Incubation, Staining, Imaging, Fluorescence