Journal: Frontiers in cell and developmental biology

Article Title: ORF3a-Mediated Incomplete Autophagy Facilitates Severe Acute Respiratory Syndrome Coronavirus-2 Replication.

doi: 10.3389/fcell.2021.716208

Figure Lengend Snippet: FIGURE 1 | Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) ORF3a triggers incomplete autophagy. (A–B) SARS-CoV-2 infection triggers incomplete autophagy. HeLa-hACE2 (A) or Vero-E6 (B) cells were infected with SARS-CoV-2 (MOI = 1) and cell lysates were collected at indicated time point post-infection for immunoblotting (IB) with indicated antibodies. (C) Screening of SARS-CoV-2-encoded proteins for LC3-I to LC3-II conversion in HeLa cells. (D,E) SARS-CoV-2 ORF3a induces incomplete autophagy. HeLa-vector and HeLa-ORF3a stable cells were treated with or without BafA1 (100 nM) for 4 h and the cell lysates were collected for IB with indicated antibodies (D). HeLa cells were transfected with increasing amount ORF3a expressing plasmid and the cell lysates were collected for IB with indicated antibodies at 48 post-transfection (E). (F) ORF3a expression modulates autophagy in Vero-E6 cells. (G) ORF3a blocks SQSTM1/p62 degradation upon starvation. HeLa-vector or HeLa-ORF3a stable cells were treated by serum starvation for 4 h and the cell lysates were collected for IB with indicated antibodies. (H,I) SARS-CoV-2 ORF3a induces LC3 puncta formation. HeLa-vector or HeLa-ORF3a cell line were treated with BafA1 (100 nM) and endogenous LC3 puncta were immunostained (H) and quantified (I). Scale bar, 15 µm. Arrow: representative autophagosomes. (J,K) HeLa-mRFP-GFP-LC3 stable cells were transfected with ORF3a or vector control and transfected cells were fixed and the LC3 puncta were captured (J,K) quantified as indicated. Yellow puncta, autophagosomes; Red puncta, autolysosomes; Arrow, representative autophagosomes. p62/GAPDH or LC3-II/GAPDH levels were quantified by the band intensity in (A–G). Similar results were obtained by three independent experiments. Mean ± SEM; n = 50; ***p < 0.001 and ****p < 0.0001 by Student’s t test in panels (I,K).

Article Snippet: For the LC3 conversion assay, SARS-CoV-2-infected or ORF3a expressing cells were washed with cold PBS, lysed with 1% Triton X-100, and then subjected to immunoblot analysis (15% SDS-PAGE) with antibodies against LC3 (Cell Signaling, #3868) or SQSTM1/p62 (Cell Signaling, #39749).

Techniques: Infection, Western Blot, Plasmid Preparation, Transfection, Expressing, Control

Journal: Frontiers in cell and developmental biology

Article Title: ORF3a-Mediated Incomplete Autophagy Facilitates Severe Acute Respiratory Syndrome Coronavirus-2 Replication.

doi: 10.3389/fcell.2021.716208

Figure Lengend Snippet: FIGURE 4 | SARS-CoV ORF3a cannot induce autophagy. (A) Sequence alignment between SARS-CoV-2 ORF3a and SARS-CoV ORF3a. (B–D) SARS-CoV ORF3a does not affect autophagy. HeLa-vector, HeLa-ORF3aSARS-CoV-2, or HeLa-ORF3aSARS-CoV cell lines were treated with BafA1 (100 nM) and endogenous LC3 puncta were immunostained (B) and quantified (C). Scale bar, 15 µm. Arrow: representative autophagosomes. Mean ± SEM; n = 50; ns and ****p < 0.0001 by one-way ANOVA and Bonferroni’s post hoc test. HeLa-vector or HeLa-ORF3aSARS-CoV cell lines were treated with BafA1 (100 nM) for 4 h and the cell lysates were collected for IB with indicated antibodies (D). (E) ORF3aSARS-CoV-2 but not ORF3aSARS-CoV interacts with endogenous UVRAG. HEK293T cells were transfected with Flag-ORF3aSARS-CoV or Flag-ORF3aSARS-CoV-2 and cell lysates were collected and subjected to IP and IB with indicated antibodies at 48h post-transfection. (F–H) SARS-CoV ORF3a does not affect the formation of UVRAG complex (F), Beclin 1 complex (G), or Atg14 complex (H). HEK293T cells were co-transfected with indicated plasmids and cell lysates were collected and subjected to IP and IB with indicated antibodies at 48 h post-transfection.

Article Snippet: For the LC3 conversion assay, SARS-CoV-2-infected or ORF3a expressing cells were washed with cold PBS, lysed with 1% Triton X-100, and then subjected to immunoblot analysis (15% SDS-PAGE) with antibodies against LC3 (Cell Signaling, #3868) or SQSTM1/p62 (Cell Signaling, #39749).

Techniques: Sequencing, Plasmid Preparation, Transfection

Journal: British journal of pharmacology

Article Title: Sodium rutin extends lifespan and health span in mice including positive impacts on liver health.

doi: 10.1111/bph.15410

Figure Lengend Snippet: FIGURE 6 Sodium rutin (NaR) treatment improves autophagy activity in hepatocytes. (a,b) Western blot analysis of the expression levels of p62 and LC3 in the liver from 24-month-old mice. (c) Transmission electron microscopy shows autophagosome (yellow arrow) in liver (scale bar = 200 nm). (d) Statistic results of autophagosome in control and NaR treatment group per cell basis (n = 6). (e–g) Representative and quantificative image of early autophagosomes (yellow foci) and autolysosomes (red foci) in murine liver with AAV-RFP-GFP-LC3 infection (n = 6, scale bar = 10 μm). (h–k) Western blot results and quantification of p62 and LC3 protein levels in HepG2 cells with different treatment (quantification data were observed from three dependent experiment, and the data normalized to the control, one-way ANOVA analysis was performed in (i) and two-way ANOVA analysis was performed in (k), (*P < .05,). (l) Oil red O staining of HepG2 cells after each treatment with metformin as a positive control (scale bar = 50 μm). (m,n) Oil red O absorbance at 520 nm and TG concentration of HepG2 cells with each treatment (NaR: 200 μM, 24 h; oleic acid: 500 μM, 12 h; chloroquine (CQ): 100 μM, 2 h; metformin: 2 mM, 4 h; Student's t-test, *P < .05,)

Article Snippet: The antibodies used were anti-SQSTM1 (Cell Signaling Technology, 5114S, 1:1000), ant- LC3 (Cell Signaling Technology, 4599S, 1:1000), anti-ANGPTL8 (Proteintech Cat# 66641-1-Ig, RRID:AB_2882000), anti-β actin (Cwbiotech, CW0096, 1:1000), GAPDH (CWBio Cat# CW0100M, RRID:AB_2801390) HRP tagged Goat anti-rabbit, (Cwbiotech, CW0103, 1:5000), HRP tagged Goat anti-mouse, (Cwbiotech, CW0102, 1:5000).

Techniques: Activity Assay, Western Blot, Expressing, Transmission Assay, Electron Microscopy, Control, Infection, Staining, Positive Control, Concentration Assay

Journal: Frontiers in Immunology

Article Title: SARS-CoV-2 Spike Targets USP33-IRF9 Axis via Exosomal miR-148a to Activate Human Microglia

doi: 10.3389/fimmu.2021.656700

Figure Lengend Snippet: Spike transfected cells released exosomes are loaded with miR-148a and miR-590. (A) Work-flow schematic depicting exosome harvesting protocol from Spike gene transfected HEK-293T cells. (B) Size distribution analysis graph of control exosomes done on NanoSight, NTA 3.2 Dev Build 3.2.16, indicating mode of size range 90.6 nm. (C) Size distribution analysis graph of S-exosomes, indicating mode of size range of 107.5 nm. (D) Immunoblot image for characterization of harvested exosome preparations. Endosomal protein Calnexin is absent in exosome population indicating the purity of exosome and free of cellular debris. TSG101 as tetraspanin marker protein are present in exosomes. (E) Graph bars showing relatively higher amount of exosome secretion by Spike transfected cells, measured as total protein via Bradford assay. (F) Graph bars displaying relative miR-590 levels in Spike-Exosomes compared to control exosomes. (G) Graph bars displaying relative levels of miR-148a in Spike-Exosomes as compared to control exosomes. Exosomes pellets were subjected to total RNA isolation and microRNA levels measured through qPCR analysis via TaqMan assay specific for miR-590 and miR-148a respectively. (H) Western blot image, confirming the Spike gene transfection in HEK-293T cells as well their accumulation in secreted exosomes. 1µg and 2 µg of Spike gene (S) plasmids (pTwist-EF1α-nCoV-2019-S-2×Strep) were transfected in 6-well plate format for 24 hours with the help of Lipofectamine 2000. (I) Western blot image showing CD63 in harvested exosomes as exosomal marker. All the experiments have been repeated at least three times independently to obtain mean and mean ± S.E.M. Levels of significance have been calculated by student’s t-test and statistical significance indicated as single * for p values <0.05 and *** for p values <0.0005.

Article Snippet: Antibodies used in this study are; anti-Calnexin, (#2679, Cell Signaling technology), anti-CD63, (#sc-5275), USP42 (sc-390604, Santacruz Biotech), USP33 (#sc-100632, Santacruz Biotech), GAPDH (#sc‐32233, Santacruz Biotech), USP7 (#D17C6, Cell Signaling Technology), TSG101 (#sc-13611, Santacruz Biotech), IRF9 (#76684, Cell Signaling Technology), SARS-CoV-2 Spike Protein S2 Monoclonal Antibody (1A9), # MA5-35946, Invitrogen).

Techniques: Transfection, Control, Western Blot, Marker, Bradford Assay, Isolation, TaqMan Assay

Journal: Cell Reports Medicine

Article Title: Truncating NFKB1 variants cause combined NLRP3 inflammasome activation and type I interferon signaling and predispose to necrotizing fasciitis

doi: 10.1016/j.xcrm.2024.101503

Figure Lengend Snippet: Truncating variants of NFKB1 impair autophagy (A) MitoSOX staining of untreated NFKB1 R157∗/R157∗ and WT THP-1 monocytes was analyzed by flow cytometry. Quantifications and representative plots are shown. (B) HMDMs (left) or NFKB1 R157∗/R157∗ and WT THP-1 monocytes (right) were activated with LPS, and MitoTempo was applied 1 h before ATP. Secreted mature IL-1β was detected by ELISA. (C) ρ 0 or conventional NFKB1 R157∗/R157∗ and WT THP-1 monocytes were activated with LPS and secreted mature IL-1β was detected by ELISA. (D) HMDMs (left) or NFKB1 R157∗/R157∗ and WT THP-1 monocytes (right) were activated with LPS for indicated times and SQSTM1 was blotted from lysates. Quantifications, representative blots, and TPL are shown. (E) NFKB1 R157∗/R157∗ and WT THP-1 monocytes were inhibited with E-64d and pepstatin A (abbreviated as EP) 1 h prior to LPS (for indicated times). LC3 was blotted from cell lysates. Quantifications, representative blot, and TPL are shown. (F and G) NFKB1 R157∗/R157∗ and WT THP-1 monocytes were treated with LPS and (F) 3-MA (6 h 45 min) or (G) rapamycin (17 h 45 min) and ATP (for the last 45 min). Secreted mature IL-1β was detected by ELISA and is expressed as fold change relative to the respective cells stimulated with LPS and ATP. (H) NFKB1 R157∗/R157∗ and WT THP-1 monocytes were activated with LPS (18 h). Cells staining positive for Hoechst (nuclear stain) and autophagic vesicle marker were analyzed by flow cytometry. Quantitation and representative plots are shown. (I) Schematic representation of the effects of truncating NFKB1 mutations on macroautophagy and NLRP3 inflammasome activation. Wilcoxon test (A), one-way RM-ANOVA (B), or two-way ANOVA (C–H) followed by Dunnett’s (B), Šidak’s (D–G), or Tukey’s test (C and H). The data are shown as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (A) n = 8; (B) HMDMs: 1 control, 3 variant carriers, THP-1 n = 4; (C–E) THP-1 n = 4; (D) HMDMs: 2 controls, 4 variant carriers; (F–H) THP-1 n = 4. See also Figure S4 .

Article Snippet: For staining with LC3 antibody (Novus Biologicals NB100-2331) THP-1 monocytes were centrifuged and lyzed in sucrose lysis buffer (250 mM Sucrose, 1 mM EDTA, and 1x protease inhibitor).

Techniques: Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Marker, Quantitation Assay, Activation Assay, Control, Variant Assay

Journal: Cell Reports Medicine

Article Title: Truncating NFKB1 variants cause combined NLRP3 inflammasome activation and type I interferon signaling and predispose to necrotizing fasciitis

doi: 10.1016/j.xcrm.2024.101503

Figure Lengend Snippet:

Article Snippet: For staining with LC3 antibody (Novus Biologicals NB100-2331) THP-1 monocytes were centrifuged and lyzed in sucrose lysis buffer (250 mM Sucrose, 1 mM EDTA, and 1x protease inhibitor).

Techniques: Control, Recombinant, Lysis, Protease Inhibitor, Western Blot, Cell Culture, Molecular Weight, Staining, Sequencing, Modification, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Transfection, Quantitation Assay, Mutagenesis, Software, CRISPR, Variant Assay, Cytometry