transfection reagent  (TransGen biotech co)

Journal: Journal of Virology

Article Title: PEDV Nsp14 induces mitophagy-mediated degradation of MAVS to antagonize host innate immunity and facilitate viral proliferation

doi: 10.1128/jvi.00498-25

Figure Lengend Snippet: PEDV Nsp14 inhibits IFN-β production. ( A ) IPEC-J2 cells were infected with or without 0.2 multiplicity of infection (MOI) PEDV, and then transfected with 5 µg/mL poly (I:C) at 18 hpi. The cells were collected at 24, 36, and 48 hpi, and the IFN-β mRNA levels were evaluated by RT-qPCR. The cells without PEDV infection and poly (I:C) transfection served as a negative control. ( B ) Each HA-tagged PEDV protein expression vector and HA-tagged empty vector (the pCAGGS-HA plasmid) were transfected separately into IPEC-J2 cells. After transfection for 36 h, protein expression was analyzed using WB. Asterisks marked the target proteins. ( C ) Each HA-tagged PEDV protein expression vector and HA-tagged empty vector were transfected separately into IPEC-J2 cells for 36 h. Following poly (I:C) transfection, RT-qPCR was used to assess IFN-β mRNA levels. The cells transfected with the HA-tagged empty vector alone served as a negative control. ( D ) IPEC-J2 cells were transfected with either the pCAGGS-HA-Nsp14 plasmid or HA-tagged empty vector. After transfection for 18 h, the cells were transfected with or without 5 µg/mL poly (I:C). The cells were collected at the indicated time points (24, 36, and 48 hpt). The IFN-β mRNA levels were assessed using RT-qPCR. ( E ) IPEC-J2 cells were transfected with gradient concentrations of the pCAGGS-HA-Nsp14 plasmid (0.5, 1, and 2 µg) or HA-tagged empty vector, followed by poly (I:C) transfection, and RT-qPCR was used to assess IFN-β mRNA levels. ( F ) IPEC-J2 cells were transfected with either the pCAGGS-HA-Nsp14 plasmid or HA-tagged empty vector. At 24 hpt, the cells were infected with or without 0.4 MOI PEDV for an additional 12 h. The IFN-β mRNA levels were assessed using RT-qPCR. The data from three independent experiments were presented as means ± SEM. Statistical analysis was conducted with one-way ANOVA or Student’s t test. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: TransIntro PL transfection reagent (FT301-01) was purchased from TransGen Biotech (Beijing, China).

Techniques: Infection, Transfection, Quantitative RT-PCR, Negative Control, Expressing, Plasmid Preparation

Journal: Journal of Virology

Article Title: PEDV Nsp14 induces mitophagy-mediated degradation of MAVS to antagonize host innate immunity and facilitate viral proliferation

doi: 10.1128/jvi.00498-25

Figure Lengend Snippet: PEDV Nsp14 inhibits IFN-β production by degrading MAVS via the autolysosomal pathway. ( A ) IPEC-J2 cells were co-transfected with either the pCAGGS-HA-Nsp14 plasmid or HA-tagged empty vector, and the Flag-tagged empty vector (the pCMV-3 × Flag-Neo plasmid), MDA5-Flag, RIG-I-Flag, MAVS-Flag, TBK1-Flag, or IRF3-Flag plasmid for 36 h. Subsequently, the levels of IFN-β mRNA were assessed using RT-qPCR. The cells co-transfected with HA-tagged empty vector and the Flag-tagged empty vector served as controls. ( B ) and ( C ) IPEC-J2 cells were transfected with either the pCAGGS-HA-Nsp14 plasmid or HA-tagged empty vector. The cells were collected at the indicated time points (24, 36, and 48 hpt). The MAVS mRNA and protein levels were detected by RT-qPCR and WB, respectively. ( D ) IPEC-J2 cells were transfected with the HA-tagged empty vector and gradient concentrations of pCAGGS-HA-Nsp14 plasmid (0.5, 1, and 2 µg) for 36 h and analyzed using WB. ( E ) IPEC-J2 cells were transfected with the pCAGGS-HA-Nsp14 plasmid or HA-tagged empty vector. After 36 h of transfection, the IP assay was performed with anti-HA magnetic beads, and WB was conducted with the specific antibodies. ( F ) The cytotoxicity of 3-MA, CQ, BafA1, or MG132 on IPEC-J2 cells was detected using the cell viability assay. ( G ) The pCAGGS-HA-Nsp14 plasmid or HA-tagged empty vector was transfected into IPEC-J2 cells. At 6 hpt, the cells were treated with 3-MA (10 mM), CQ (50 µM), BafA1 (200 nM), or MG132 (5 µM) for an additional 30 h. WCLs were analyzed by WB. ( H ) IPEC-J2 cells were co-transfected with the GFP-LC3 plasmid and the pCAGGS-HA-Nsp14 plasmid or HA-tagged empty vector. At 6 hpt, the cells were treated with CQ for an additional 30 h. The cells were subjected to confocal microscopy using the specific antibodies and fluorescent reagents. The assessment of co-localization was conducted by calculating Manders’ overlap coefficient using the JaCoP plugin in the ImageJ software. Scale bars = 10 µm. Data represent means ± SEM from three independent experiments. Statistical analysis was carried out using Student’s t test. ns P > 0.05 and **** P < 0.0001.

Article Snippet: TransIntro PL transfection reagent (FT301-01) was purchased from TransGen Biotech (Beijing, China).

Techniques: Transfection, Plasmid Preparation, Quantitative RT-PCR, Magnetic Beads, Viability Assay, Confocal Microscopy, Software

Journal: Journal of Virology

Article Title: PEDV Nsp14 induces mitophagy-mediated degradation of MAVS to antagonize host innate immunity and facilitate viral proliferation

doi: 10.1128/jvi.00498-25

Figure Lengend Snippet: PEDV Nsp14 induces mitophagy-mediated degradation of MAVS to inhibit IFN-β production and facilitate viral proliferation during infection in IPEC-J2 cells. ( A, B ) IPEC-J2 cells were infected with or without 0.2 MOI PEDV and collected at the indicated time points (24, 36, and 48 hpi). The MAVS mRNA and protein levels were detected by RT-qPCR and WB, respectively. ( C through G ) IPEC-J2 cells were transfected with siNC, siNsp14, or siNDP52. At 24 hpt, the cells were infected with 0.2 MOI PEDV. In parallel, Mdivi-1 was applied as a control. The MAVS, TOM20, and PEDV N protein abundance were analyzed by WB ( C ). After poly (I:C) transfection, the mRNA levels of IFN-β were detected by RT-qPCR. The cells transfected with siNC alone served as control ( D ). RT-qPCR was used to evaluate the PEDV mRNA levels ( E ). PEDV infectivity was detected using IFA with the mouse anti-PEDV N protein mAb. The cells were stained with DAPI for nuclear staining. Scale bars = 50 µm ( F ). PEDV titers were measured by assessing TCID 50 ( G ). Data represent means ± SEM from three independent experiments. Statistical analysis was carried out using one-way ANOVA or Student’s t test. ns P > 0.05, *** P < 0.001 and **** P < 0.0001.

Article Snippet: TransIntro PL transfection reagent (FT301-01) was purchased from TransGen Biotech (Beijing, China).

Techniques: Infection, Quantitative RT-PCR, Transfection, Control, Quantitative Proteomics, Staining