Journal: Frontiers in Microbiology

Article Title: Autophagy Induced by the N-Terminus of the Classic Swine Fever Virus Nonstructural Protein 5A Protein Promotes Viral Replication

doi: 10.3389/fmicb.2021.733385

Figure Lengend Snippet: Nonstructural protein 5A expression induces cellular autophagy. (A) PK-15 cells were transfected for NS5A expression, infected with CSFV (MOI=1) for 36h or treated with rapamycin (Rapa, 100nM) for 48h. (B-D) The expression of light chain 3 (LC3), ATG5, BECN1, and GAPDH (loading control) was analyzed by immunoblotting with specific antibodies described in the Materials and Methods. (E) PK-15 cells were transfected with NS5A or treated with Rapa (100nM) for 48h. The cells were then fixed and processed for indirect immunofluorescence using antibodies against LC3B, followed by the corresponding secondary antibodies conjugated to FITC as described in the Materials and Methods. The cell nuclei were counterstained with DAPI. The fluorescence signals were visualized by confocal immunofluorescence microscopy. In the images, nuclear staining is shown in blue, and LC3B staining is shown in green. (F) The average number of LC3B puncta in each cell was determined from at least 100 cells in each group. The data represent the mean±SD of three independent experiments. Statistical comparisons were evaluated using ANOVA followed by Tukey’s HSD post hoc analysis. *** p <0.001, ** p <0.01, and compared with the mock group.

Article Snippet: The primary antibodies were as follows: anti-LC3B (Abcam, ab-229327), anti-ATG5 (Bioss, bs-4005R), anti-Beclin 1 (Bioss, bs-1353R), and anti-GAPDH (Bioss, bs-0755R).

Techniques: Expressing, Transfection, Infection, Western Blot, Immunofluorescence, Fluorescence, Microscopy, Staining

Journal: Frontiers in Microbiology

Article Title: Autophagy Induced by the N-Terminus of the Classic Swine Fever Virus Nonstructural Protein 5A Protein Promotes Viral Replication

doi: 10.3389/fmicb.2021.733385

Figure Lengend Snippet: Expression of autophagy marker proteins in cells expressing different portions of the NS5A gene. (A) PK-15 cells were transfected with sequences expressing different portions of the NS5A gene or a mock plasmid. The expression of LC3, ATG5, BECN1, and GAPDH (loading control) was analyzed by immunoblotting with specific antibodies described in the Materials and Methods. The data represent the mean±SD of three independent experiments. Statistical comparisons were evaluated using ANOVA followed by Tukey’s HSD post hoc analysis. *** p <0.001, compared with the mock group.

Article Snippet: The primary antibodies were as follows: anti-LC3B (Abcam, ab-229327), anti-ATG5 (Bioss, bs-4005R), anti-Beclin 1 (Bioss, bs-1353R), and anti-GAPDH (Bioss, bs-0755R).

Techniques: Expressing, Marker, Transfection, Plasmid Preparation, Western Blot

Journal: Frontiers in Microbiology

Article Title: Autophagy Induced by the N-Terminus of the Classic Swine Fever Virus Nonstructural Protein 5A Protein Promotes Viral Replication

doi: 10.3389/fmicb.2021.733385

Figure Lengend Snippet: The key amino acids of NS5A affect the expression of autophagy marker proteins. (A) PK-15 cells were transfected with constructs expressing NS5A with specific amino acid substitutions. The expression of LC3, ATG5, BECN1, and GAPDH (loading control) was analyzed by immunoblot with specific antibodies described in the Materials and Methods. The data represent the mean±SD of three independent experiments. Statistical comparisons were evaluated using ANOVA followed by Tukey’s HSD post hoc analysis. *** p <0.001, ** p <0.01, compared with the mock group.

Article Snippet: The primary antibodies were as follows: anti-LC3B (Abcam, ab-229327), anti-ATG5 (Bioss, bs-4005R), anti-Beclin 1 (Bioss, bs-1353R), and anti-GAPDH (Bioss, bs-0755R).

Techniques: Expressing, Marker, Transfection, Construct, Western Blot

Journal: Journal of Virology

Article Title: Swine MicroRNAs ssc-miR-221-3p and ssc-miR-222 Restrict the Cross-Species Infection of Avian Influenza Virus

doi: 10.1128/JVI.01700-20

Figure Lengend Snippet: AIV infection and ssc-miR-221-3p/222 transfection induce PAM cell apoptosis. (A and B) PAM cells were infected with AIV (H5N1, H7N9, and H9N2) as indicated at an MOI of 1 for 24 h. (C and D) PAM cells were transfected with miRNA mimics or control at 60 nM for 24 h. The cells were collected by centrifugation and were resuspended in 100 μl of 1× binding buffer supplemented with 5 μl of PE annexin V and 5 μl 7-AAD. Fluorescence of the stained cells was then analyzed using flow cytometry. The total protein was collected and Bcl2, caspase 3, and cleave caspase 3 protein levels were determined by Western blotting, with β-actin as an internal control. Data are presented as means ± SD of results from three independent experiments. Statistical significance was based on two-way ANOVA (**, P < 0.01; ***, P < 0.001).

Article Snippet: Primary antibodies included anti-β-actin (1:1,000; AA128; Beyotime), anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH; 1:1,000; AF0006; Beyotime), anti-NP (WB, 1:1,000; A01506; GenScript), anti-NF-κB P65 (WB, 1:1,000; ChIP, 1:100; 6956; Cell Signaling Technology), anti-phospho-NF-κB P65 (Ser536) (1:1,000; 3033; Cell Signaling Technology), anti-IκBα (1:1,000; 4814; Cell Signaling Technology), anti-phospho-IκBα (Ser32) (1:1,000; bsm-52169R; Bioss), anti-HMBOX1 (1:1,000; bs-18047R; Bioss), anti-Bcl2 (1:1,000; bsm-33047M; Bioss), anti-caspase 3/P17/P19 (1:1,000; 19677-1-AP; Proteintech), and anti-NP (IFA, 1:5,000; ab20343; Abcam).

Techniques: Infection, Transfection, Centrifugation, Binding Assay, Fluorescence, Staining, Flow Cytometry, Western Blot

Journal: Journal of Virology

Article Title: Swine MicroRNAs ssc-miR-221-3p and ssc-miR-222 Restrict the Cross-Species Infection of Avian Influenza Virus

doi: 10.1128/JVI.01700-20

Figure Lengend Snippet: ssc-miR-221-3p and ssc-miR-222 target HMBOX1. (A) Selection of host targets of ssc-miR-221-3p/222. HEK293T cells were cotransfected with luciferase reporter vectors pGL3 containing the seven potential target genes, pRL-TK, and 80 nM miRNA mimics or 80 nM randomized oligonucleotide as a mimic control. Cell lysates were harvested for dual-luciferase assays at 24 hpi. (B) Alignments of HMBOX1 binding sites with miR-221-3p/222. Perfect matches are indicated by a line between the 3′ UTR of HMBOX1 (HMBOX1-wt) and miR-221-3p/222. Mutations (in red in HMBOX1-mut) were made in the seed region of the miR-221-3p/222 binding site for the reporter assay. 293T cells were cotransfected with 80 nM ssc-miR-221-3p/222 mimic or randomized oligonucleotide, the reporter plasmid HMBOX1 or HMBOX1-mut, and pRL-TK. Cell lysates were harvested for dual-luciferase assays at 24 hpi. (C) Conserved miR-221-3p/222 binding sites in the 3′ UTR of HMBOX1. (D) ssc-miR-221-3p/222 inhibited HMBOX1 expression in PAM cells. PAM cells were transfected with miRNA controls or miR-221-3p/222 mimics at 80 nM. At 24 h after transfection, cells were harvested, and the cytosolic proteins were examined by Western blotting using anti-HMBOX1 polyclonal antibodies. GAPDH expression was used as an internal control. (E) PAM cells were transfected with 60 nM HMBOX1-specific siRNA (siHMBOX1) or scrambled siRNA (siCtrl) as a control for 24 h, followed by H5N1 AIV infection (MOI, 1) for 24 h. PAM cells were harvested for Western blotting against HMBOX1, NP, Bcl2, caspase 3, and cleave caspase 3, with β-actin as an internal control. Data are presented as means ± SD of results from three independent experiments. Statistical significance was based on two-way ANOVA (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Article Snippet: Primary antibodies included anti-β-actin (1:1,000; AA128; Beyotime), anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH; 1:1,000; AF0006; Beyotime), anti-NP (WB, 1:1,000; A01506; GenScript), anti-NF-κB P65 (WB, 1:1,000; ChIP, 1:100; 6956; Cell Signaling Technology), anti-phospho-NF-κB P65 (Ser536) (1:1,000; 3033; Cell Signaling Technology), anti-IκBα (1:1,000; 4814; Cell Signaling Technology), anti-phospho-IκBα (Ser32) (1:1,000; bsm-52169R; Bioss), anti-HMBOX1 (1:1,000; bs-18047R; Bioss), anti-Bcl2 (1:1,000; bsm-33047M; Bioss), anti-caspase 3/P17/P19 (1:1,000; 19677-1-AP; Proteintech), and anti-NP (IFA, 1:5,000; ab20343; Abcam).

Techniques: Selection, Luciferase, Binding Assay, Reporter Assay, Plasmid Preparation, Expressing, Transfection, Western Blot, Infection