Fig 1: RTN3 knockdown enhances antiviral immune responses.(A) RT-PCR analysis (top) and immunoblotting analysis (bottom) of RTN3 mRNA levels in PBMCs infected with Ctrl or RTN3 shRNA encoding lentivirus (shCtrl, shRNA #1, shRNA #2) for 12 hr and cultured for additional 24 hr (top). (B) Immunoblotting analys of PBMCs infected with Ctrl or RTN3 shRNA encoding lentivirus for 12 hr and cultured for another 24 hr, followed by infection with VSV-eGFP (MOI = 2) at the indicated timepoints (left). Quantitative comparison of the indicated protein levels was analyzed by gray intensity scanning of blots and is shown (right). (C–D) RT-PCR analysis of IFNB1, IFIT2, IFIT1, TNF, IL6, and CCL20 mRNA levels (C) and VSV viral genomic RNA mRNA levels (D) in PBMCs infected with Ctrl or RTN3 shRNA encoding lentivirus for 12 hr and cultured for another 24 hr, followed by infection with VSV-eGFP (MOI = 2) at the indicated timepoints. In (B), the data are representative of three independent experiments. In (A, C, D), the data are shown as the mean values ± SD (n = 3). *, p < 0.0332; **, p < 0.0021; ***, p < 0.0002; and ****, p < 0.0001 by Sidak’s multiple comparisons test. Figure 3—source data 1.Uncropped blots with bands labeled AI file and raw unedited blots files for panels A and B. Figure 3—source data 2.Excel file of RT-PCR measurements and description of statistical tests for panel A. Figure 3—source data 3.Excel file of blots quantification and description of statistical tests for panel B. Figure 3—source data 4.Excel file of RT-PCR measurements and description of statistical tests for panel C. Figure 3—source data 5.Excel file of RT-PCR measurements and description of statistical tests for panel D.
Fig 2: RTN3 is upregulated and self-aggregates upon RNA viral infection.(A) Immunoblot analysis of HEK293T cells infected with VSV-eGFP (MOI = 1) or treated with poly(I:C) (5 µg/ml) at the indicated timepoints. (B–C) mRNA levels of RTN3 and TNF in the same samples shown in (A) were detected by real-time PCR. B. VSV-eGFP-infected group, C. poly(I:C)-treated group. (D) Immunoblot analysis of HEK293T cells treated with TNF-a (10 ng/ml) or IFN-ß (20 ng/ml) at the indicated timepoints. (E) mRNA levels of RTN3 in the same samples shown in (D) were detected by RT-PCR. (F) Immunoblot analysis of PBMCs infected with VSV-eGFP (MOI = 2) at the indicated timepoints (top), and mRNA levels of RTN3 in the same samples were detected by RT-PCR (bottom). In (A, D, F), the data are representative of three independent experiments. In (B, C, E, F), the data are shown as the mean values ± SD (n = 3). *, p < 0.0332; **, p < 0.0021; ***, p < 0.0002; and ****, p < 0.0001 by Sidak’s multiple comparisons test. Figure 1—source data 1.Uncropped blots with bands labeled AI file and raw unedited blots files for panels A, D, and F. Figure 1—source data 2.Excel file of RT-PCR measurements and description of statistical tests for panel B. Figure 1—source data 3.Excel file of RT-PCR measurements and description of statistical tests for panel C. Figure 1—source data 4.Excel file of RT-PCR measurements and description of statistical tests for panel E. Figure 1—source data 5.Excel file of RT-PCR measurements and description of statistical tests for panel F.
Fig 3: RTN3 overexpression suppresses antiviral immune responses.(A) Luciferase assays of HEK293T cells transfected with an ISRE luciferase reporter (ISRE-Luc), an IFNB luciferase reporter (IFNß-Luc) or an NF-?B luciferase reporter (NF-?B-Luc) together with an HA-tagged empty vector (HA-Ev, no wedge) or increasing amounts (wedge) of plasmid encoding RTN3 (HA-RTN3) followed by transfection with a plasmid encoding the RIG-I CARD domain [RIG-I (N)] to activate the pathway. Cell lysates were collected 24 hr posttransfection, the same as that described in the following experiments, if no additional annotation was performed. (B) Luciferase activity of HEK293T cells transfected with ISRE-Luc, together with HA-Ev or increasing amounts of plasmid HA-RTN3, followed by treatment with poly(I:C) (5 µg/ml) or SeV (MOI = 0.1) for 16 or 24 hr. (C) Fluorescence and phase contrast (PH) analyses of HEK293T cells transfected with HA-Ev or HA-RTN3, followed by infection with VSV-eGFP (MOI = 0.05) at the indicated timepoints (left). Scale bar, 100 µm. mRNA levels of VSV viral RNA in the same samples were analyzed by RT-PCR (right). (D) The percentage of eGFP-positive cells in the same samples shown in (C) was analyzed by flow cytometry. (E) Immunoblot analysis of HEK293T cells transfected with HA-Ev or HA-RTN3 followed by infection with VSV-eGFP (MOI = 1) at the indicated timepoints (left). Quantitative comparison of the indicated protein levels analyzed by gray intensity scanning of blots (right). (F) RT-PCR analysis of IFNB1, IFIT2, IFIT1, TNF, IL6, and CCL20 mRNA levels in HEK293T cells transfected with HA-Ev or HA-RTN3 followed by infection with VSV-eGFP (MOI = 1) at the indicated timepoints. In (E), the data are representative of three independent experiments. In (A, B, C, F), the data are shown as the mean values ± SD (n = 3). *, p < 0.0332; **, p < 0.0021; ***, p < 0.0002; and ****, p < 0.0001 by Sidak’s multiple comparisons test. Figure 2—source data 1.Uncropped blots with bands labeled AI file and raw unedited blots files for panel E. Figure 2—source data 2.Excel file of luciferase reporter assay measurements and description of statistical tests for panel A. Figure 2—source data 3.Excel file of luciferase reporter assay measurements and description of statistical tests for panel B. Figure 2—source data 4.Excel file of RT-PCR measurements and description of statistical tests for panel C. Figure 2—source data 5.Excel file of blots quantification and description of statistical tests for panel E. Figure 2—source data 6.Excel file of RT-PCR measurements and description of statistical tests for panel F.
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