Journal: Frontiers in Microbiology

Article Title: A Host Factor GPNMB Restricts Porcine Circovirus Type 2 (PCV2) Replication and Interacts With PCV2 ORF5 Protein

doi: 10.3389/fmicb.2018.03295

Figure Lengend Snippet: Both PCV2 and ORF5 reduced the expression of GPNMB in PAMs. (A) PCR detection of PCV2 nucleic acid in PAM cells with PCV2 infection at a MOI of 1.0 at 24 and 48 h post-infection. Marker. DNA Marker DL1000. (B) Real-time qRT-PCR analysis of GPNMB mRNA expression in PAM cells infected with PCV2 a MOI of 1 at indicated time points. (C) Immunoblot analysis of GPNMB protein expression in PAM cells infected with PCV2 a MOI of 1 at indicated time points. (D) Immunoblot analysis of GFP protein expression in PAM cells transfected with GFP-C1 or GFP ORF5 construct at 48 h post-transfection. (E) Real-time qRT-PCR analysis of GPNMB mRNA expression in PAM cells transfected with GFP-C1 or GFP ORF5 construct at indicated time points. (F) Immunoblot analysis of GPNMB protein expression in PAM cells transfected with GFP-C1 or GFP ORF5 construct at indicated time points. Data are shown as the mean ± SD of three independent experiments and measured in technical duplicates in (B) and (E) . Data were normalized to housekeeping gene β-actin expression. Comparisons between groups were determined with the Student’s t -test. ∗ p < 0.05; ∗∗∗ p < 0.001.

Article Snippet: Membranes were blocked in TBST, a blocking buffer containing 5% skim milk for 2 h at room temperature, followed by incubation with primary antibodies, including rabbit anti-GPNMB polyclonal antibody, rabbit anti-Cyclin A polyclonal antibody (1:1000, Santa Cruz Biotechnology, United States), and rabbit anti-β-actin polyclonal antibody (1:2000, CWBIO, China) at 4°C for overnight.

Techniques: Expressing, Infection, Marker, Quantitative RT-PCR, Western Blot, Transfection, Construct

Journal: Frontiers in Microbiology

Article Title: A Host Factor GPNMB Restricts Porcine Circovirus Type 2 (PCV2) Replication and Interacts With PCV2 ORF5 Protein

doi: 10.3389/fmicb.2018.03295

Figure Lengend Snippet: Interaction of ORF5 with GPNMB. (A) Exogenous co-IP analysis of ORF5 and GPNMB in PAMs. Cells were co-transfected with plasmids GFP-ORF5 and Flag-GPNMB. PAMs co-transfected with GFP-C1 and Flag-GPNMB were used as negative controls. A quarter of the cell extract was subjected to the input assay to assess β-actin, Flag-fusion and GFP-fusion protein levels. The rest of the extract was subjected to IP assay. Western blot detected proteins with a mouse anti-GFP mAb and a mouse anti-Flag pAb. (B) ORF5 protein co-localizes with GPNMB. HEK-293T cells were co-transfected with pGPNMB-Red and pORF5-GFP, pEGFP-C1 and pDsRed-N1 were used as control. Cells were fixed and stained with DAPI (blue) at 48 h post-transfection. Scale bar = 10 μm. (C) Endogenous co-IP analysis of ORF5 and GPNMB in PAMs. Cells were transfected with plasmid GFP-ORF5-Flag and GFP-C1-transfected PAMs were used as negative controls. The input assay was performed using a quarter of the cell extract to assess β-actin, Flag fusion protein and GPNMB levels. (D) GST-ORF5 pull-down assay. The GST and GST-ORF5 proteins expressed in Escherichia coli Rosetta (DE3) cells were immobilized on a glutathione agarose resin, followed by incubation of the resin with the cell lysates containing GPNMB-Flag protein.

Article Snippet: Membranes were blocked in TBST, a blocking buffer containing 5% skim milk for 2 h at room temperature, followed by incubation with primary antibodies, including rabbit anti-GPNMB polyclonal antibody, rabbit anti-Cyclin A polyclonal antibody (1:1000, Santa Cruz Biotechnology, United States), and rabbit anti-β-actin polyclonal antibody (1:2000, CWBIO, China) at 4°C for overnight.

Techniques: Co-Immunoprecipitation Assay, Transfection, Western Blot, Staining, Plasmid Preparation, Pull Down Assay, Incubation

Journal: Frontiers in Microbiology

Article Title: A Host Factor GPNMB Restricts Porcine Circovirus Type 2 (PCV2) Replication and Interacts With PCV2 ORF5 Protein

doi: 10.3389/fmicb.2018.03295

Figure Lengend Snippet: Stable overexpression of GPNMB inhibits PCV2 replication and ORF5 expression. (A) Real-time qRT-PCR analysis of GPNMB mRNA expression in PK-15 cells with stable GPNMB overexpression. (B) Immunoblot analysis of GPNMB protein in PK-15 cells with stable GPNMB overexpression. The right panel is the GPNMB protein level that the intensity of the signal for targeted protein were normalized to that from β-actin with three independent experiments. (C,D) Real-time qRT-PCR analysis of PCV2 viral RNA (C) and ORF5 mRNA (D) expression in PK-15 cells with stable GPNMB overexpression. Different cell lines were infected with PCV2 at a MOI of 0.1. PCV2 viral RNA level was measured 24 h or 48 h post-infection. Data are shown as the mean ± SD of three independent experiments and measured in technical duplicates in panels A , C , and D . Data were normalized to housekeeping gene β-actin expression. Comparisons between groups were determined with the Student’s t -test. ∗ p < 0.05; ∗∗ p < 0.01.

Article Snippet: Membranes were blocked in TBST, a blocking buffer containing 5% skim milk for 2 h at room temperature, followed by incubation with primary antibodies, including rabbit anti-GPNMB polyclonal antibody, rabbit anti-Cyclin A polyclonal antibody (1:1000, Santa Cruz Biotechnology, United States), and rabbit anti-β-actin polyclonal antibody (1:2000, CWBIO, China) at 4°C for overnight.

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Western Blot, Infection

Journal: Frontiers in Microbiology

Article Title: A Host Factor GPNMB Restricts Porcine Circovirus Type 2 (PCV2) Replication and Interacts With PCV2 ORF5 Protein

doi: 10.3389/fmicb.2018.03295

Figure Lengend Snippet: Transient overexpression of Red-fused GPNMB inhibits PCV2 replication and ORF5 expression. (A) Real-time qRT-PCR analysis of GPNMB mRNA expression in PK-15 cells transfected with pDsRed-GPNMB or PdsRed-N1 at 24 h post-transfection. (B) Immunoblot analysis of GPNMB protein in PK-15 cells transfected with pDsRed-GPNMB or PdsRed-N1 at 24 h post-transfection. The right panel is the GPNMB protein level that the intensity of the signal for targeted protein were normalized to that from β-actin with three independent experiments. (C,D) Real-time qRT-PCR analysis of PCV2 viral RNA (C) and ORF5 mRNA (D) expression in PK-15 cells transfected with pDsRed-GPNMB or PdsRed-N1. Different cells were infected with PCV2 at a MOI of 0.1 at 24 h post-transfection. RNA expression level was measured at 24 h post-infection. Data are shown as the mean ± SD of three independent experiments and measured in technical duplicates in panels A , C , and D . Data were normalized to housekeeping gene β-actin expression. Comparisons between groups were determined with the Student’s t -test. ∗ p < 0.05; ∗∗ p < 0.01.

Article Snippet: Membranes were blocked in TBST, a blocking buffer containing 5% skim milk for 2 h at room temperature, followed by incubation with primary antibodies, including rabbit anti-GPNMB polyclonal antibody, rabbit anti-Cyclin A polyclonal antibody (1:1000, Santa Cruz Biotechnology, United States), and rabbit anti-β-actin polyclonal antibody (1:2000, CWBIO, China) at 4°C for overnight.

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Transfection, Western Blot, Infection, RNA Expression

Journal: Frontiers in Microbiology

Article Title: A Host Factor GPNMB Restricts Porcine Circovirus Type 2 (PCV2) Replication and Interacts With PCV2 ORF5 Protein

doi: 10.3389/fmicb.2018.03295

Figure Lengend Snippet: Knockdown of GPNMB increases PCV2 replication and ORF5 expression. (A) Real-time qRT-PCR analysis of GPNMB mRNA expression in stable GPNMB knockdown PK-15 cells. PK-15 cells transduced with GPNMB-knockdown lentivirus (shGPNMB-1, 2, 3) or random sequence control (shN). (B) Immunoblot analysis of GPNMB protein in stable GPNMB knockdown PK-15 cells. The right panel is the GPNMB protein level that the intensity of the signal for targeted protein were normalized to that from β-actin with three independent experiments. (C,D) Real-time qRT-PCR analysis of PCV2 viral RNA (C) and ORF5 mRNA (D) expression in stable GPNMB knockdown. Different cells were infected with PCV2 at a MOI of 0.1. RNA expression level was measured at 24 h or 48 h post-infection. Data are shown as the mean ± SD of three independent experiments and measured in technical duplicates in panels A , C , and D . Data were normalized to housekeeping gene β-actin expression. Comparisons between groups were determined with the Student’s t -test. ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: Membranes were blocked in TBST, a blocking buffer containing 5% skim milk for 2 h at room temperature, followed by incubation with primary antibodies, including rabbit anti-GPNMB polyclonal antibody, rabbit anti-Cyclin A polyclonal antibody (1:1000, Santa Cruz Biotechnology, United States), and rabbit anti-β-actin polyclonal antibody (1:2000, CWBIO, China) at 4°C for overnight.

Techniques: Expressing, Quantitative RT-PCR, Transduction, Sequencing, Western Blot, Infection, RNA Expression

Journal: Frontiers in Microbiology

Article Title: A Host Factor GPNMB Restricts Porcine Circovirus Type 2 (PCV2) Replication and Interacts With PCV2 ORF5 Protein

doi: 10.3389/fmicb.2018.03295

Figure Lengend Snippet: Overexpression of GPNMB promotes cell cycle progression. (A) Real-time qRT-PCR analysis of Cyclin A mRNA expression in PAM cells transfected with pEGFP-C1 or pEGFP-ORF5 construct at 24 h or 48 h post-transfection. (B) Immunoblot analysis of Cyclin A protein expression in PAM cells transfected with pEGFP-C1 or pEGFP-ORF5 construct at 24 h or 48 h post-transfection. (C,D) Real-time qRT-PCR analysis of GPNMB (C) and Cyclin A (D) mRNA expression in PAMs with stable GPNMB overexpression. (E) Immunoblot analysis of GPNMB and Cyclin A protein levels in PAM cells with stable GPNMB overexpression. The middle panel is the GPNMB protein level and the right panel is the Cyclin A protein level that the intensity of the signal for targeted protein were normalized to that from β-actin with three independent experiments. (F) Histograms from flow cytometry data for propidium iodide (PI) staining for the mock cells (Mock), control lentivirus transduced cells (Lenti) and GPNMB overexpression lentivirus transduced cells (Lenti-GPNMB). Data are shown as the mean ± SD of three independent experiments and measured in technical duplicates in panels A and B . Data were normalized to housekeeping gene β-actin expression. Comparisons between groups were determined with the Student’s t -test. ∗ p < 0.05; ∗∗ p < 0.01.

Article Snippet: Membranes were blocked in TBST, a blocking buffer containing 5% skim milk for 2 h at room temperature, followed by incubation with primary antibodies, including rabbit anti-GPNMB polyclonal antibody, rabbit anti-Cyclin A polyclonal antibody (1:1000, Santa Cruz Biotechnology, United States), and rabbit anti-β-actin polyclonal antibody (1:2000, CWBIO, China) at 4°C for overnight.

Techniques: Over Expression, Quantitative RT-PCR, Expressing, Transfection, Construct, Western Blot, Flow Cytometry, Staining

Journal: Frontiers in Microbiology

Article Title: A Host Factor GPNMB Restricts Porcine Circovirus Type 2 (PCV2) Replication and Interacts With PCV2 ORF5 Protein

doi: 10.3389/fmicb.2018.03295

Figure Lengend Snippet: Knockdown of GPNMB inhibits cell cycle progression. (A,B) Real-time qRT-PCR analysis of GPNMB mRNA (A) and Cyclin A mRNA (B) expression in PAMs cell lines with GPNMB knockdown. (C) Immunoblot analysis of GPNMB and Cyclin A protein levels in PAM cells with lines with GPNMB knockdown. The middle panel is the GPNMB protein level and the right panel is the Cyclin A protein level that the intensity of the signal for targeted protein were normalized to that from β-actin with three independent experiments. (D) Histograms from flow cytometry data for propidium iodide (PI) staining for the mock cells (Mock), control lentivirus transduced cells (shN) and GPNMB knockdown lentivirus transduced cells (shGPNMB-3). Data are shown as the mean ± SD of three independent experiments and measured in technical duplicates in panels A and B . Data were normalized to housekeeping gene β-actin expression. Comparisons between groups were determined with the Student’s t -test. ∗ p < 0.05.

Article Snippet: Membranes were blocked in TBST, a blocking buffer containing 5% skim milk for 2 h at room temperature, followed by incubation with primary antibodies, including rabbit anti-GPNMB polyclonal antibody, rabbit anti-Cyclin A polyclonal antibody (1:1000, Santa Cruz Biotechnology, United States), and rabbit anti-β-actin polyclonal antibody (1:2000, CWBIO, China) at 4°C for overnight.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Flow Cytometry, Staining