lc3 conversion assay  (Cell Signaling Technology Inc)

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