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: Cell reports

Article Title: TRPML1 Promotes Protein Homeostasis in Melanoma Cells by Negatively Regulating MAPK and mTORC1 Signaling

doi: 10.1016/j.celrep.2019.07.086

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: DQ-BSA Green , Fisher Scientific , Cat# D12050.

Techniques: Virus, Generated, Recombinant, Reverse Transcription, Saline, Cloning, Gel Extraction, Western Blot, Stripping, Bicinchoninic Acid Protein Assay, shRNA, Software, SDS-Gel