Journal: Frontiers in Cardiovascular Medicine

Article Title: MAGI1 inhibits interferon signaling to promote influenza A infection

doi: 10.3389/fcvm.2022.791143

Figure Lengend Snippet: MAGI1 suppresses IFN signaling in ECs. (A) HUVECs were transfected with either siCont or siMAGI1. After 48 h of transfection, total RNA was extracted then qRT-PCR was performed. Relative changes in Magi1, Mx1, Ifnb1, Ifna1, IfngI, Stat1, Stat5a, and Stat5b expression were calculated using the comparative C t ( 2−ΔΔCt ) method. Ct values of each target gene were normalized to that of the Gapdh . Each group passed the Shapiro-Wilk normality test, then an unpaired student t -test was performed using the Prism software (GraphPad Software). The graph shows mean ± SD ( n = 11). ** p < 0.01, * p < 0.05. (B) HUVECs were transfected with either siCont or siMAGI1. After 48 h of transfection, total RNA was extracted then qRT-PCR was performed. Relative changes in Oas2 mRNA expression were calculated using the comparative C t ( 2−ΔΔCt ) method. Ct values of Oas2 were normalized to that of the Gapdh . Each group passed the Shapiro-Wilk normality test, then an unpaired student t -test was performed using the Prism software (GraphPad Software). The graph shows mean ± SD ( n = 3). ** p < 0.01. (C) HUVECs were transfected with either siCont or siMAGI1. After 48 h of transfection, cell lysates were analyzed by immunoblotting with each specific antibody as indicated. The graphs represent densitometry data from 3 independent gels, one of which is shown in the left panel. Each group passed the Shapiro-Wilk normality test, then an unpaired student t -test was performed using the Prism software (GraphPad Software). The graph shows mean ± SD, n = 3, ** P < 0.01, and * P < 0.05. Uncropped figures were provided in the supplements. (D) HUVECs were transfected with either siCont or siMAGI1. After 48 h of transfection, conditioned medium was collected and levels of IFNβ were measured. Each group passed the Shapiro-Wilk normality test, then an unpaired student t -test was performed using the Prism software (GraphPad Software). The graph shows mean ± SD, n = 4–6, * P < 0.05. (E,F) HUVECs were transfected with either the pCMV-MAGI1 or the pCMV-2B backbone construct (control) . After 24 h of transfection, total RNA was extracted then qRT-PCR was performed. Relative changes in Magi1 (E) and Ifnb1 (F) mRNA expression were calculated using the comparative C t ( 2−ΔΔCt ) method. Ct values of each target gene were normalized to that of the gapdh . At least, one of the groups did not pass the Shapiro-Wilk normality test, we performed an unpaired t -test with Welch's correction using the Prism software (GraphPad Software). The graph shows mean ± SD, n = 5, ** P < 0.01. (G) HUVECs were transfected with either the pCMV-MAGI1 or the pCMV-2B backbone construct (control) . After 24 h of transfection, the cells were treated with IFN (200 U/mL) for 6 h, and immunoblotting was performed with each specific antibodies as indicated. The graph (lower) represents densitometry data from 4 independent gels, one of which is shown in the top panel. Each group passed the Shapiro-Wilk normality test, then one-way ANOVA followed by Turkey's multiple comparisons test was performed using the Prism software (GraphPad Software). The graph shows mean ± SD ( n = 4). * p < 0.05.

Article Snippet: Authenticated siRNA sequence targeting human MX1 was obtained from Santa Cruz Biotechnology (Santa Cruz, Dallas, TX, USA).

Techniques: Transfection, Quantitative RT-PCR, Expressing, Software, Western Blot, Construct, Control

Journal: Frontiers in Cardiovascular Medicine

Article Title: MAGI1 inhibits interferon signaling to promote influenza A infection

doi: 10.3389/fcvm.2022.791143

Figure Lengend Snippet: MX1 depletion reverses MAGI1 depletion-mediated suppression of IAV infection. (A,B) HUVECs were transfected with either siCont, siMX1, siMAGI1, or siMX1+siMAGI1. After 48 h of transfection, cell lysates were analyzed by immunoblotting with each specific antibody as indicated. Gels are representative of 3 independent experiments. (C) HUVECs were transfected with either siCont, siMX1, siMAGI1, or siMX1+siMAGI1. After 48 h of transfection, the cells were infected with IAV for 24 h. Total RNA was extracted then qRT-PCR was performed. Relative changes in the viral NP mRNA expression were calculated using the comparative C t ( 2−ΔΔCt ) method. Ct values of the viral NP were normalized to that of the 18S ribosomal RNA . Each group passed the Shapiro-Wilk normality test, then one-way ANOVA followed by Turkey's multiple comparisons test was performed using the Prism software (GraphPad Software). The graph shows mean ± SD ( n = 3-4). ** p < 0.01, * p < 0.05. (D) Mouse lung ECs were isolated from Magi1 −/− and littermate wild type (WT) control mice and cultured. The cells were infected with IAV for 24h. Total RNA was extracted then qRT-PCR was performed. Relative changes in the viral NP mRNA expression were calculated using the comparative C t ( 2−ΔΔCt ) method. Ct values of the viral NP were normalized to that of the 18S ribosomal RNA . Each group passed the Shapiro-Wilk normality test, then an unpaired student t -test was performed using the Prism software (GraphPad Software). The graph shows mean ± SD, n =11. NS: not significant.

Article Snippet: Authenticated siRNA sequence targeting human MX1 was obtained from Santa Cruz Biotechnology (Santa Cruz, Dallas, TX, USA).

Techniques: Infection, Transfection, Western Blot, Quantitative RT-PCR, Expressing, Software, Isolation, Control, Cell Culture

Journal: Frontiers in Cardiovascular Medicine

Article Title: MAGI1 inhibits interferon signaling to promote influenza A infection

doi: 10.3389/fcvm.2022.791143

Figure Lengend Snippet: MAGI1 depletion promotes IRF3 nuclear translocation and de-SUMOylation without affecting IRF3 S396 phosphorylation. (A) HUVECs were transfected with either siCont or siMAGI1. After 48 h of transfection, cell lysates were analyzed by immunoblotting with each specific antibody as indicated. The graphs represent densitometry data from 3 independent gels, one of which is shown in . Each group passed the Shapiro-Wilk normality test, then an unpaired student t -test was performed using the Prism software (GraphPad Software). The graphs show mean ± SD, n = 3, ** P < 0.01. (B) HUVECs were transfected with either siCont or siMAGI1. After 48 h of transfection, immunofluorescence staining for IRF3 protein (green) and DAPI staining for the nuclei (blue) were performed. Scale bars: 20 μM. Robust IRF3 nuclear translocation noted in siMAGI1-transfected cells. The percentages of cells with nuclear staining were quantified by counting the cells with the mean intensity of IRF3 staining in the nucleus ≥ mean intensity of IRF3 cytoplasmic staining by each dish ( n = 5–6) in a blinded manner. (C) HUVECs were transfected with either siCont or siMAGI1. After 48 h of transfection, cell lysates were immuno-precipitated IRF3 with the antibody that specifically recognizes this protein then immunoblotting with the antibody that recognizes SUMO1 to detect IRF3 SUMOylation . IgG antibody was used as a negative control. Total cell lysates were immunoblotted with each specific antibody as indicated. (D) The graphs represent densitometry data from 3 independent gels, one of which is shown in shown in (C) . Median intensities of IRF3 SUMOylation were calculated after subtracting the background, which is median intensities of IgG control. Each group passed the Shapiro-Wilk normality test, then an unpaired student t -test with Welch's correction was performed using the Prism software (GraphPad Software). The graph shows mean ± SD, n = 3, ** P < 0.01. (E) The scheme depicts the relationship between MAGI1 and IFN signaling. IAV infection induces anti-viral responses including STATs and IRF3 activation and subsequent MX1 and OAS2 induction. For efficient infection of IAV to ECs, IAV induces MAGI1 expression, resulting in inhibition of anti-viral responses by inhibiting STATs and IRF3 activation. Especially, MAGI1 inhibits IRF3 de-SUMOylation and subsequent IRF3 activation. Importantly, OxLDL also upregulates MAGI1 expression and promotes IAV infection, which may explain the relationship between hypercholesterolemia and IAV infection in patients. The scheme was generated using BioRender.

Article Snippet: Authenticated siRNA sequence targeting human MX1 was obtained from Santa Cruz Biotechnology (Santa Cruz, Dallas, TX, USA).

Techniques: Translocation Assay, Phospho-proteomics, Transfection, Western Blot, Software, Immunofluorescence, Staining, Negative Control, Control, Infection, Activation Assay, Expressing, Inhibition, Generated

Journal: Journal of Virology

Article Title: Porcine Mx1 Protein Inhibits Classical Swine Fever Virus Replication by Targeting Nonstructural Protein NS5B

doi: 10.1128/JVI.02147-17

Figure Lengend Snippet: Porcine Mx depletion enhances CSFV replication. (A to D) Efficiency of siMx1, siMx2, siGBP1–765, or siISG15 in PK-15 cells. Western blotting was performed on lysates from PK-15 cells treated with 100 nM siMx1, siMx2, siGBP1–765, siISG15, or siNC for 12 h and then treated with 100 ng of huIFN-α for 36 h. poMx1, poMx2, GBP-1, ISG15, and GAPDH were detected using mouse monoclonal antibodies against poMx1, poMx2, GBP-1, ISG15, and GAPDH, respectively. (E) Effects of Mx1, Mx2, GBP-11–765, or ISG15 on CSFV replication. PK-15 cells pretreated with siRNAs were treated with 100 ng of huIFN-α for 36 h and then infected with CSFV at an MOI of 0.1 for 48 h. Viral RNA levels, virus titers, and viral E2 levels were determined by RT-qPCR, TCID50, and Western blotting, respectively. Results are presented as the means ± SD of data from three independent experiments. **, P < 0.01.

Article Snippet: Two specific siRNAs targeting different regions of the Mx1 and Mx2 genes (sc-45260 and sc61110, respectively) and a nontargeting control siRNA ([siNC] sc-37007) were purchased from Santa Cruz Biotechnology, Inc.; ISG15 and GBP-1 genes were commercially synthesized by GenePharma (Shanghai, China). siRNA duplexes used in the study are listed in .

Techniques: Western Blot, Bioprocessing, Infection, Virus, Quantitative RT-PCR