Journal: Frontiers in Immunology

Article Title: The STING/TBK1/IRF3/IFN type I pathway is defective in cystic fibrosis

doi: 10.3389/fimmu.2023.1093212

Figure Lengend Snippet: cGAS/STING pathway is downregulated in ΔF BMDMs (A) Western blot analysis of TBK1, IRF3 and p-IRF3 expression levels in WT and ΔF BMDMs after 4h and 6h infection with Pseudomonas aeruginosa strain (PAO-1). Actin was used as loading control. (B) Densitometric analysis showing p-IRF3 and actin expression levels in BMDMs following PAO-1 infection (n=3; means ± SEM; ***p<0.001). (C) IFN-β mRNA levels, quantified by qPCR, in BMDMs obtained from WT and ΔF mice after 4h and 6h infection with PAO-1, normalized to the mRNA levels of actin. (n=3; means ± SEM; ***p<0.001). (D) Western blot analysis of TBK1 and IRF3, and their phosphorylated forms, with their respective densitometric analysis (E, F) , in PAO-1 infected WT, HE and ΔF BMDMs following overnight cysteamine treatment. Actin was used to normalize protein loading levels. (n=3; means ± SEM; *p<0.05; ***p<0.001). (G) IFN-β mRNA expression levels, quantified by qPCR, in BMDMs treated with cysteamine, and infected with PAO-1 for 4h. Actin mRNA levels were used to normalize IFN-β mRNA levels. (n=3; means ± SEM; **p<0.01; ***p<0.001).

Article Snippet: Primary antibodies used: Anti-IRF3 (D83B9) Cat# 4302 (Cell Signaling); anti-phospho- IRF3 (Ser396) (D601M) Cat#29047 (Cell Signaling); anti-STAT1 (D1K9Y) Cat# 14994 (Cell Signaling); anti-phospho-STAT1 (Tyr701) (58D6) Cat#9167 (Cell Signaling): anti- STING (D2P2F) Cat# 13647 (Cell Singaling); anti-TBK1/NAK (D1B4) Cat#3504 (Cell Signaling); anti-phospho-TBK1/NAK (Ser172) (D52C2) Cat#5483 (Cell Signaling); Anti-Actin (Sigma).

Techniques: Western Blot, Expressing, Infection

Journal: Frontiers in Immunology

Article Title: The STING/TBK1/IRF3/IFN type I pathway is defective in cystic fibrosis

doi: 10.3389/fimmu.2023.1093212

Figure Lengend Snippet: 2’,3’ cGAMP restores the STING axis in ΔF ex vivo models (A) Western blot analysis of TBK1 and IRF3, and their phosphorylated forms (B) , with their respective densitometric analysis (C, D) , in PAO-1 infected WT and ΔF BMDMs, following overnight cysteamine treatment and 2h of 2’,3’ cGAMP stimulation. Actin was used to normalize protein loading levels. (n=3; means ± SEM; *p<0.05; ***p<0.001). (E) IFN-β mRNA levels, quantified by qPCR, in WT and ΔF BMDMs treated with cysteamine and 2’,3’cGAMP. Actin mRNA levels were used to normalize IFN-β mRNA levels (n=3; means ± SEM; ***p<0.001). (F) PAO-1 internalization at T=10 expressed as number of CFUs. (G) Living PAO-1 after 4 hours expressed as number of CFUs. (H) Percentage of PAO-1 clearance expressed as living bacteria after 4 hours of culture with respect to internalized bacteria. (n=3; means ± SEM; ***p<0.001). (I) IFN-β mRNA levels, quantified by qPCR, in Human PBMCs from ΔF (n=4) and healthy donors (HD) (n=4). Cells were stimulated with 2’,3’ cGAMP for 1h and then infected with PAO-1 for 2h. The mRNA levels of IFN-β were normalized to the mRNA levels of actin. (**p<0.01).

Article Snippet: Primary antibodies used: Anti-IRF3 (D83B9) Cat# 4302 (Cell Signaling); anti-phospho- IRF3 (Ser396) (D601M) Cat#29047 (Cell Signaling); anti-STAT1 (D1K9Y) Cat# 14994 (Cell Signaling); anti-phospho-STAT1 (Tyr701) (58D6) Cat#9167 (Cell Signaling): anti- STING (D2P2F) Cat# 13647 (Cell Singaling); anti-TBK1/NAK (D1B4) Cat#3504 (Cell Signaling); anti-phospho-TBK1/NAK (Ser172) (D52C2) Cat#5483 (Cell Signaling); Anti-Actin (Sigma).

Techniques: Ex Vivo, Western Blot, Infection

Journal: Frontiers in Immunology

Article Title: The STING/TBK1/IRF3/IFN type I pathway is defective in cystic fibrosis

doi: 10.3389/fimmu.2023.1093212

Figure Lengend Snippet: 2’,3’ cGAMP restores the STING axis increasing bacterial removal in ΔF mice (A) Western blot analysis of IRF3, TBK1, STAT1 and their phosphorylated forms, with their respective densitometric analysis, in the lungs of WT and ΔF mice subjected to 2’,3’ cGAMP pre-treatment and to 4h PAO-1 infection. Actin was used as protein loading control. (n=4; means ± SEM; *p<0.05; **p<0.01). (B) IFN-β mRNA levels, quantified by qPCR, in lungs from PAO-1 infected in WT and ΔF mice following 2’,3’ cGAMP pre-treatment for 2h and then PAO-1 infected for 4h. The mRNA levels of IFN- β were normalized to the mRNA levels of actin. (n=4; means ± SEM; *p<0.05). (C) IFN-β protein expression quantified by ELISA, in lungs from PAO-1 infected in WT and ΔF mice following 2’,3’ cGAMP pre-treatment for 2h and then PAO-1 infected for 4h. (n=4; means ± SEM; *p<0.05; **p<0.01). (D) Enumeration of living PAO-1 bacteria in lung after 4h of infection expressed as number of CFUs (n=4; means ± SEM; ***p<0.001).

Article Snippet: Primary antibodies used: Anti-IRF3 (D83B9) Cat# 4302 (Cell Signaling); anti-phospho- IRF3 (Ser396) (D601M) Cat#29047 (Cell Signaling); anti-STAT1 (D1K9Y) Cat# 14994 (Cell Signaling); anti-phospho-STAT1 (Tyr701) (58D6) Cat#9167 (Cell Signaling): anti- STING (D2P2F) Cat# 13647 (Cell Singaling); anti-TBK1/NAK (D1B4) Cat#3504 (Cell Signaling); anti-phospho-TBK1/NAK (Ser172) (D52C2) Cat#5483 (Cell Signaling); Anti-Actin (Sigma).

Techniques: Western Blot, Infection, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: The STING/TBK1/IRF3/IFN type I pathway is defective in cystic fibrosis

doi: 10.3389/fimmu.2023.1093212

Figure Lengend Snippet: cGAS/STING pathway is downregulated in ΔF BMDMs (A) Western blot analysis of TBK1, IRF3 and p-IRF3 expression levels in WT and ΔF BMDMs after 4h and 6h infection with Pseudomonas aeruginosa strain (PAO-1). Actin was used as loading control. (B) Densitometric analysis showing p-IRF3 and actin expression levels in BMDMs following PAO-1 infection (n=3; means ± SEM; ***p<0.001). (C) IFN-β mRNA levels, quantified by qPCR, in BMDMs obtained from WT and ΔF mice after 4h and 6h infection with PAO-1, normalized to the mRNA levels of actin. (n=3; means ± SEM; ***p<0.001). (D) Western blot analysis of TBK1 and IRF3, and their phosphorylated forms, with their respective densitometric analysis (E, F) , in PAO-1 infected WT, HE and ΔF BMDMs following overnight cysteamine treatment. Actin was used to normalize protein loading levels. (n=3; means ± SEM; *p<0.05; ***p<0.001). (G) IFN-β mRNA expression levels, quantified by qPCR, in BMDMs treated with cysteamine, and infected with PAO-1 for 4h. Actin mRNA levels were used to normalize IFN-β mRNA levels. (n=3; means ± SEM; **p<0.01; ***p<0.001).

Article Snippet: Primary antibodies used: Anti-IRF3 (D83B9) Cat# 4302 (Cell Signaling); anti-phospho- IRF3 (Ser396) (D601M) Cat#29047 (Cell Signaling); anti-STAT1 (D1K9Y) Cat# 14994 (Cell Signaling); anti-phospho-STAT1 (Tyr701) (58D6) Cat#9167 (Cell Signaling): anti- STING (D2P2F) Cat# 13647 (Cell Singaling); anti-TBK1/NAK (D1B4) Cat#3504 (Cell Signaling); anti-phospho-TBK1/NAK (Ser172) (D52C2) Cat#5483 (Cell Signaling); Anti-Actin (Sigma).

Techniques: Western Blot, Expressing, Infection

Journal: Frontiers in Immunology

Article Title: The STING/TBK1/IRF3/IFN type I pathway is defective in cystic fibrosis

doi: 10.3389/fimmu.2023.1093212

Figure Lengend Snippet: 2’,3’ cGAMP restores the STING axis in ΔF ex vivo models (A) Western blot analysis of TBK1 and IRF3, and their phosphorylated forms (B) , with their respective densitometric analysis (C, D) , in PAO-1 infected WT and ΔF BMDMs, following overnight cysteamine treatment and 2h of 2’,3’ cGAMP stimulation. Actin was used to normalize protein loading levels. (n=3; means ± SEM; *p<0.05; ***p<0.001). (E) IFN-β mRNA levels, quantified by qPCR, in WT and ΔF BMDMs treated with cysteamine and 2’,3’cGAMP. Actin mRNA levels were used to normalize IFN-β mRNA levels (n=3; means ± SEM; ***p<0.001). (F) PAO-1 internalization at T=10 expressed as number of CFUs. (G) Living PAO-1 after 4 hours expressed as number of CFUs. (H) Percentage of PAO-1 clearance expressed as living bacteria after 4 hours of culture with respect to internalized bacteria. (n=3; means ± SEM; ***p<0.001). (I) IFN-β mRNA levels, quantified by qPCR, in Human PBMCs from ΔF (n=4) and healthy donors (HD) (n=4). Cells were stimulated with 2’,3’ cGAMP for 1h and then infected with PAO-1 for 2h. The mRNA levels of IFN-β were normalized to the mRNA levels of actin. (**p<0.01).

Article Snippet: Primary antibodies used: Anti-IRF3 (D83B9) Cat# 4302 (Cell Signaling); anti-phospho- IRF3 (Ser396) (D601M) Cat#29047 (Cell Signaling); anti-STAT1 (D1K9Y) Cat# 14994 (Cell Signaling); anti-phospho-STAT1 (Tyr701) (58D6) Cat#9167 (Cell Signaling): anti- STING (D2P2F) Cat# 13647 (Cell Singaling); anti-TBK1/NAK (D1B4) Cat#3504 (Cell Signaling); anti-phospho-TBK1/NAK (Ser172) (D52C2) Cat#5483 (Cell Signaling); Anti-Actin (Sigma).

Techniques: Ex Vivo, Western Blot, Infection

Journal: Frontiers in Immunology

Article Title: The STING/TBK1/IRF3/IFN type I pathway is defective in cystic fibrosis

doi: 10.3389/fimmu.2023.1093212

Figure Lengend Snippet: 2’,3’ cGAMP restores the STING axis increasing bacterial removal in ΔF mice (A) Western blot analysis of IRF3, TBK1, STAT1 and their phosphorylated forms, with their respective densitometric analysis, in the lungs of WT and ΔF mice subjected to 2’,3’ cGAMP pre-treatment and to 4h PAO-1 infection. Actin was used as protein loading control. (n=4; means ± SEM; *p<0.05; **p<0.01). (B) IFN-β mRNA levels, quantified by qPCR, in lungs from PAO-1 infected in WT and ΔF mice following 2’,3’ cGAMP pre-treatment for 2h and then PAO-1 infected for 4h. The mRNA levels of IFN- β were normalized to the mRNA levels of actin. (n=4; means ± SEM; *p<0.05). (C) IFN-β protein expression quantified by ELISA, in lungs from PAO-1 infected in WT and ΔF mice following 2’,3’ cGAMP pre-treatment for 2h and then PAO-1 infected for 4h. (n=4; means ± SEM; *p<0.05; **p<0.01). (D) Enumeration of living PAO-1 bacteria in lung after 4h of infection expressed as number of CFUs (n=4; means ± SEM; ***p<0.001).

Article Snippet: Primary antibodies used: Anti-IRF3 (D83B9) Cat# 4302 (Cell Signaling); anti-phospho- IRF3 (Ser396) (D601M) Cat#29047 (Cell Signaling); anti-STAT1 (D1K9Y) Cat# 14994 (Cell Signaling); anti-phospho-STAT1 (Tyr701) (58D6) Cat#9167 (Cell Signaling): anti- STING (D2P2F) Cat# 13647 (Cell Singaling); anti-TBK1/NAK (D1B4) Cat#3504 (Cell Signaling); anti-phospho-TBK1/NAK (Ser172) (D52C2) Cat#5483 (Cell Signaling); Anti-Actin (Sigma).

Techniques: Western Blot, Infection, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: TRAF3 activates STING-mediated suppression of EV-A71 and target of viral evasion

doi: 10.1038/s41392-022-01287-2

Figure Lengend Snippet: EV-A71 infection triggers STING-mediated immune activation and viral suppression. a Relative expression of STING and cGAS in HEK-293T and THP-1 cells. Cells were cultured and harvested for western blot analysis. STING and cGAS levels were evaluated using anti-STING and anti-cGAS antibodies. GAPDH was used as a loading control. b , c STING was efficiently down-regulated in shSTING-THP-1 cells compared with control cells and was quantified using ImageJ software (National Institutes of Health, Maryland, USA). Cells were cultured and harvested for western blot analysis. STING was tested using an anti-STING antibody. GAPDH was used as a loading control. d STING could be activated in control THP-1 cells and such activation was impaired when STING was down-regulated. Equal numbers of THP-1 control or shSTING cells were seeded into 12-well plates and treated with 100 nM STING agonist for 12 h and harvested for western blot analysis. MDA5, DDX58, p-TBK1 (Ser172), p-IRF3 (Ser386), ISG60, ISG56, ISG54, STING, p-STING (Ser366), RSAD2, ISG15 and GAPDH were probed using the indicated antibodies. GAPDH was used as a loading control. e – h EV-A71 infection could efficiently induce STING-triggered down-stream gene activation. Equal volumes of EV-A71 (MOI of 0.5) were used to infect control/shSTING-THP-1 cells (2 × 10 5 /well in 24-well plates). Viral preservation solution was used as a negative control. 24 h later, cells were harvested for western blot analysis. MDA5, DDX58, p-TBK1 (Ser172), p-IRF3 (Ser386), ISG60, ISG56, ISG54, STING, p-STING (Ser366), RSAD2, ISG15, VP1 and GAPDH were probed using the indicated antibodies. GAPDH was used as a loading control. p-STING (Ser366) ( f ), p-TBK1 (Ser172) ( g ), and p-IRF3 (Ser386) ( h ) protein expressions were represented by western blot bands and were quantified using ImageJ software. n = 3 independent experiments were performed (representative immunoblots are shown). i Inhibition of EV-A71-stimulated human genes by silencing STING expression. THP-1 control or shSTING cells were infected with EV-A71 for 24 h and harvested for total RNA extraction, first strand cDNA synthesis, and RT-qPCR analysis with indicated interferon stimulated genes specific primers. Human genes activated by EV-A71 alone served as a positive control and were set to 100%. Total RNA was prepared from THP-1 cells and analyzed for the transcriptional level of the indicated genes by RT-qPCR ( n = 3 independent biological experiments). Statistical significance was determined by two-sided unpaired t -test. j Representative images of JC-1-stained THP-1 cells. Cells were infected with EV-A71 for 24 h and subjected to mitochondrial membrane potential using JC-1 staining. Scale bar: 80 µm. k HEK-293T STING stable cell line were transfected with cGAS expression vectors or empty vectors. After 36 h, those transfected cells were then mock-infected or infected with EV-A71 at an MOI of 0.5 for 4 h and harvested for western blot analysis. p-TBK1 (Ser172), ISG60, cGAS, STING, p-STING (Ser366), ISG15, and Histone H3 were probed using the indicated antibodies. Histone H3 was used as a loading control. Data in a – k represent the average of results from three independent experiments ( n = 3, representative immunoblots are shown). Error bars indicate the standard deviation of the data from three independent experiments. Means and standard deviations are presented. Statistical significance was determined using two-sided unpaired t -test, * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: Anti-Phospho-TBK1/NAK (Ser172) (rabbit / IgG, Cat No. #5483), anti-Phospho-IRF-3 (Ser396) (rabbit mAb, Cat No. #4947), anti-TBK1/NAK (rabbit mAb, Cat No. #38066), and anti-IRF-3 (rabbit mAb, Cat No. #11904), anti-Phospho-STING (rabbit / IgG, Cat No.50907 s) antibodies were acquired from Cell Signaling Technology (Massachusetts, USA).

Techniques: Infection, Activation Assay, Expressing, Cell Culture, Western Blot, Software, Preserving, Negative Control, Inhibition, RNA Extraction, Quantitative RT-PCR, Positive Control, Staining, Stable Transfection, Transfection, Standard Deviation

Journal: Signal Transduction and Targeted Therapy

Article Title: TRAF3 activates STING-mediated suppression of EV-A71 and target of viral evasion

doi: 10.1038/s41392-022-01287-2

Figure Lengend Snippet: EV-A71 infection inhibits cGAS-STING pathway activation. a HEK-293T cells were infected with EV-A71 (MOI of 0.5) or mock-infected with virus preservation solution. Twenty-two hours later, cells were transfected with IRF3 , NF-κB , IL-8 , IFNβ , or ISRE promoter, together with cGAS-STING expression vectors or empty vector. Eighteen hours post-transfection, cells were harvested for western blot analysis. Cell lysates were separated by SDS-PAGE, transferred to PVDF membranes, and incubated with the indicated antibodies to detect p-TBK1 (Ser172), p-IRF3 (Ser386), ISG60, ISG56, ISG54, RSAD2, EV-A71 VP1, p-STING (Ser366), ISG15 and GAPDH. Anti-myc and anti-Flag antibodies were used to detect cGAS and STING expression. GAPDH was used as a loading control. b–f . cGAS-STING-stimulated promoters were suppressed following EV-A71 infection. Eighteen hours post transfection ( a ), IRF3 ( b ) , NF-κB ( c ) , IL-8 (d) , IFN-β ( e ), and ISRE ( f ) promoter luciferase activity was measured using a dual-luciferase reporter gene assay. Luciferase induced by cGAS-STING served as a control and was set to 100%. pRL-TK Renilla was used as an internal control. g HEK-293T cells were infected with EV-A71 (MOI of 0.5) or mock-infected with virus preservation solution. Twenty-two hours later, cells were transfected with cGAS-STING expression vectors or empty vector. Eighteen hours post-transfection, cells were harvested for total RNA extraction. First strand cDNA synthesis and RT-qPCR analysis with indicated interferon stimulated genes specific primers. Human genes activated by cGAS-STING transfection served as a positive control and were set to 100%. Total RNA was prepared from 293 T cells and analyzed for the transcriptional level of the indicated genes by RT-qPCR. Data in a – g represent the average of results from three independent experiments ( n = 3, representative immunoblots are shown). Error bars indicate the standard deviation of the data from three independent experiments. The means and standard deviations are presented. Statistical significance was determined using a two-sided unpaired Student’s t -test, * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: Anti-Phospho-TBK1/NAK (Ser172) (rabbit / IgG, Cat No. #5483), anti-Phospho-IRF-3 (Ser396) (rabbit mAb, Cat No. #4947), anti-TBK1/NAK (rabbit mAb, Cat No. #38066), and anti-IRF-3 (rabbit mAb, Cat No. #11904), anti-Phospho-STING (rabbit / IgG, Cat No.50907 s) antibodies were acquired from Cell Signaling Technology (Massachusetts, USA).

Techniques: Infection, Activation Assay, Preserving, Transfection, Expressing, Plasmid Preparation, Western Blot, SDS Page, Incubation, Luciferase, Activity Assay, Reporter Gene Assay, RNA Extraction, Quantitative RT-PCR, Positive Control, Standard Deviation

Journal: Signal Transduction and Targeted Therapy

Article Title: TRAF3 activates STING-mediated suppression of EV-A71 and target of viral evasion

doi: 10.1038/s41392-022-01287-2

Figure Lengend Snippet: 2A pro EV-A71 inhibits cGAS-STING pathway activation. a Schematic diagram of the genomic organization and encoded proteins of EV-A71 strain. b , c EV-A71 2A pro rather than other viral proteins suppressed cGAS-STING-induced IFN-β and NF-κB promoter activation. HEK-293T cells were co-transfected with IFN-β ( b ) or NF-κB ( c ) promoter, empty vector, or cGAS-STING expressing vectors, in the presence or absence of EV-A71 VP0, VP1, VP2, VP3, VP4, 2 A, 2B, 2 C, 3 A, 3B, 3 C and 3D expressing vectors. Transactivation of the luciferase reporter gene was determined 18 h after transfection. Luciferase activity induced by cGAS-STING served as a control and was set to 100%. pRL-TK Renilla was used as an internal control. d – f EV-A71 2A pro inhibited cGAS-STING-induced IRF3, IL-8 and ISRE promoter activation. HEK-293T cells were co-transfected with IRF3 (D), IL-8 (E) or ISRE (F) promoters, empty vector, or cGAS-STING-expressing vectors, in the presence or absence of EV-A71 2A pro expressing vectors. Eighteen hours post-transfection, cells were harvested for dual-luciferase reporter gene assays. g EV-A71 2A pro inhibited cGAS-STING triggered TBK1, IRF3 phosphorylation and ISG60 activation. HEK-293T cells were co-transfected with empty vector, cGAS-STING-expressing vectors, empty vector, or EV-A71 2A pro expressing vector. Eighteen hours post-transfection, cells were harvested for western blot analysis. Anti-myc, anti-Flag, and anti-HA antibodies were used to detect cGAS, STING, and EV-A71 2A pro expression, respectively. p-TBK1 (Ser172), p-IRF3 (Ser386), ISG60, and GAPDH were probed using the indicated antibodies. GAPDH was used as a loading control. h – m Enzymatic active site of EV-A71 2A pro appears to be important for its inhibitory activity against cGAS-STING function. HEK-293T cells were co-transfected with IRF3 ( h ), NF-κB ( i ), IL-8 ( j ), IFN-β ( k ) or ISRE ( l ) promoters, empty vector, or cGAS-STING-expressing vectors, empty vector, or EV-A71 2A pro wild type (WT) or EV-A71 2A pro C110A expressing vectors. Transactivation of the luciferase reporter gene was determined 18 h after transfection. Luciferase activity induced by cGAS-STING served as a control and was set to 100%. pRL-TK Renilla was used as an internal control. m HEK-293T cells were co-transfected with empty vector, cGAS-STING-expressing vectors, empty vector, or EV-A71 2A pro WT or C110A expressing vector. Eighteen hours post-transfection, cells were harvested for western blot analysis. Anti-HA antibodies were used to detect EV-A71 2A pro expression. ISG60, STING, p-STING (Ser366), ISG15 and GAPDH were probed using the indicated antibodies. GAPDH was used as a loading control. Data in b – m represent the average of results from three independent experiments ( n = 3, representative immunoblots are shown). The error bars indicate the standard deviations of data from three independent experiments. Means and standard deviations are presented. Statistical significance was determined by two-sided unpaired Student’s t -test, ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: Anti-Phospho-TBK1/NAK (Ser172) (rabbit / IgG, Cat No. #5483), anti-Phospho-IRF-3 (Ser396) (rabbit mAb, Cat No. #4947), anti-TBK1/NAK (rabbit mAb, Cat No. #38066), and anti-IRF-3 (rabbit mAb, Cat No. #11904), anti-Phospho-STING (rabbit / IgG, Cat No.50907 s) antibodies were acquired from Cell Signaling Technology (Massachusetts, USA).

Techniques: Activation Assay, Transfection, Plasmid Preparation, Expressing, Luciferase, Activity Assay, Western Blot

Journal: Signal Transduction and Targeted Therapy

Article Title: TRAF3 activates STING-mediated suppression of EV-A71 and target of viral evasion

doi: 10.1038/s41392-022-01287-2

Figure Lengend Snippet: TRAF3 is a target of EV-A71 2A pro . a Schematic diagram of the process of cGAS-STING activation and factors involved. b HEK-293T cells were transfected with empty vector or EV-A71 2A pro- expressing vectors. Eighteen hours post-transfection, cells were harvested for western blot analysis. Anti-HA was used to identify EV-A71 2A pro . GAPDH was used as a loading control. c HEK-293T cells were transfected with empty vector or EV-A71 2A pro -expressing vectors ( b ), cGAS, and STING expressing vectors. Eighteen hours post-transfection, cells were harvested for western blot analysis. cGAS, STING, TBK1, IRF3, p50, p65, IKKβ, TRAF6, TRAF3 and GAPDH were detected using the indicated antibodies. GAPDH was used as a loading control. TRAF3 cleavage by EV-A71 2A pro is indicated by an arrowhead. d HEK-293T cells were transfected with increasing doses of EV-A71 2A pro WT- or C110A-expressing vectors. Eighteen hours post-transfection, cells were harvested for western blot analysis. Anti-HA and anti-TRAF3 antibodies were used to probe EV-A71 2A pro and TRAF3, respectively. GAPDH was used as a loading control. TRAF3 cleavage by EV-A71 2A pro is indicated by an arrowhead. e , f TRAF3-WT but not TRAF3-Mut was cleaved by EV-A71 2A pro . HEK-293T cells were transfected with empty vector, TRAF3-WT, TRAF3-Mut, and EV-A71 2A pro -expressing vectors. Eighteen hours post-transfection, cells were harvested for western blot analysis. Anti-HA was used to identify EV-A71 2A pro . Anti-Flag was used to identify TRAF3. GAPDH was used as a loading control. Western blot bands were quantified using ImageJ software and the cleavage efficiencies (defined by the ratio of bind 40KDa/60KDa) of TRAF3-WT and TRAF3-Mut were shown as bar in e . g HEK-293T cells were transfected with TRAF3-WT and empty vector. Eighteen hours post-transfection, cells were infected with increasing doses of EV-A71. Forty-eight hours post-infection, cells were harvested for western blot analysis. Anti-HA and anti-TRAF3 antibodies were used to probe EV-A71 2A pro and TRAF3. Histone H3 was used as a loading control. TRAF3 cleavage by EV-A71 2A pro is indicated by an arrowhead. Data in a–g represent the averages of results from three independent experiments ( n = 3, representative immunoblots are shown)

Article Snippet: Anti-Phospho-TBK1/NAK (Ser172) (rabbit / IgG, Cat No. #5483), anti-Phospho-IRF-3 (Ser396) (rabbit mAb, Cat No. #4947), anti-TBK1/NAK (rabbit mAb, Cat No. #38066), and anti-IRF-3 (rabbit mAb, Cat No. #11904), anti-Phospho-STING (rabbit / IgG, Cat No.50907 s) antibodies were acquired from Cell Signaling Technology (Massachusetts, USA).

Techniques: Activation Assay, Transfection, Plasmid Preparation, Expressing, Western Blot, Software, Infection

Journal: Signal Transduction and Targeted Therapy

Article Title: TRAF3 activates STING-mediated suppression of EV-A71 and target of viral evasion

doi: 10.1038/s41392-022-01287-2

Figure Lengend Snippet: Silencing TRAF3 expression inhibited cGAS-STING activation and TRAF3 rescued cGAS-STING activation from 2A pro ’s suppression. a – e HEK-293T cells were transfected with control siRNA (siNC) or si TRAF3 for 24 h. Next, cells were co-transfected with IRF3 ( a ), NF-κB ( b ), IL-8 ( c ), IFNβ ( d ) or ISRE ( e ) promoters, empty vector, or cGAS-STING-expressing vectors. Transactivation of the luciferase reporter gene was determined 18 h after transfection. Luciferase activity induced by cGAS-STING served as a control and was set to 100%. pRL-TK Renilla was used as an internal control. f HEK-293T cells were transfected with control siRNA (siNC) or si TRAF3 for 24 h. Next, cells were co-transfected with empty vector or cGAS-STING-expressing vectors. Eighteen hours post-transfection, cells were harvested for western blot analysis. p-TBK1 (Ser172), p-IRF3 (Ser386), ISG60, TRAF3, p-STING (Ser366), STING and GAPDH were detected using the indicated antibodies. STING was detected by anti-Flag antibody. GAPDH was used as a loading control. g , h Silencing TRAF3 decreased the number and intensity of STING foci formation. HeLa cells were transfected with Control siRNA or si TRAF3 . Twenty-four hours later, control siRNA or si TRAF3 cells were transfected with STING-Flag. Sixteen hours post-transfection, cells were primed with 100 nM STING agonist or solvent control. Six hours post agonist addition, cells were fixed, treated with Triton-X100, and incubated in 5% FBS, and subcultured to anti-Flag and following FITC Conjugate Goat Anti-Rabbit IgG (H + L) reactivation. Images were captured using a ZEISS laser scanning confocal microscope (Zeiss LSM 900, Oberkochen, Germany). ZEISS ZEN Microscope software was used for acquisition. Percentages of foci formation cells were statistical analyzed. i Co-immunoprecipitation analysis of endogenous TRAF3 with STING-Flag. HEK-293T cells were transfected with STING-Flag or a control vector, as indicated. Cell lysates were prepared and immunoprecipitated using anti-Flag beads 36 h after transfection. Precipitated samples were separated by SDS-PAGE, transferred to polyvinylidene fluoride membranes, and reacted with anti-TRAF3 antibodies to detect potential binding of TRAF3 protein and anti-Flag antibodies to detect STING-Flag. GAPDH was used as a loading control. j The effect of knocking down TRAF3 on STING-TBK1 interaction. HEK-293T cells were transfected with control siRNA (siNC) or siTRAF3. Twenty-four hours later, HEK-293T cells were co-transfected with cGAS and STING-Flag expression vectors or control vectors for 16 h. Cell lysates were prepared and immunoprecipitated using anti-Flag beads. Precipitated samples were separated by SDS-PAGE, transferred to polyvinylidene fluoride membranes, and reacted with anti-Flag, anti-TBK1, anti-p-TBK1 (Ser172), anti-TRAF3, and anti-ISG60 antibodies to detect indicating proteins. GAPDH was used as a loading control. k – p HEK-293T cells were transfected with IRF3 ( k ), NF-κB ( l ), IL-8 ( m ), IFNβ ( n ), or ISRE ( o ) promoter and cGAS-STING expressing vectors, with empty vector or EV-A71 2A pro WT or EV-A71 2A pro C110A expression vectors, in the presence or absence of vectors expressing TRAF3, as indicated. Transactivation of the luciferase reporter gene was determined 18 h after transfection. Luciferase activity induced by cGAS-STING served as a control and was set to 100%. pRL-TK Renilla was used as an internal control. p HEK-293T cells were transfected with cGAS-STING-expressing vectors, empty vector, or EV-A71 2A pro in the presence or absence of vectors expressing TRAF3, as indicated. Eighteen hours post-transfection, cells were harvested for western blot analysis. Cell lysates were separated by SDS-PAGE, transferred to PVDF membranes, and treated with antibodies to detect p-IRF3 (Ser386), ISG60, cGAS, and STING. Anti-Flag antibody was used to detect TRAF3. Anti-HA antibody was used to detect EV-A71 2A pro . GAPDH was used as a loading control. Data in b – m represent the average of results from three independent experiments ( n = 3, representative immunoblots are shown). The error bars indicate the standard deviations of data from three independent experiments. Means and standard deviations are presented. Statistical significance was determined by two-sided unpaired Student’s t -test, ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: Anti-Phospho-TBK1/NAK (Ser172) (rabbit / IgG, Cat No. #5483), anti-Phospho-IRF-3 (Ser396) (rabbit mAb, Cat No. #4947), anti-TBK1/NAK (rabbit mAb, Cat No. #38066), and anti-IRF-3 (rabbit mAb, Cat No. #11904), anti-Phospho-STING (rabbit / IgG, Cat No.50907 s) antibodies were acquired from Cell Signaling Technology (Massachusetts, USA).

Techniques: Expressing, Activation Assay, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Western Blot, Incubation, Microscopy, Software, Immunoprecipitation, SDS Page, Binding Assay

Journal: PLOS Pathogens

Article Title: Suppression of cGAS- and RIG-I-mediated innate immune signaling by Epstein-Barr virus deubiquitinase BPLF1

doi: 10.1371/journal.ppat.1011186

Figure Lengend Snippet: (A) Functional screening for IFN-β antagonists. HEK293 cells were transfected with a Firefly luciferase reporter plasmid driven by IFN-β-promoter (IFNB-Luc), a control Renilla luciferase reporter plasmid driven by thymidine kinase promoter (TK-Luc) serving to normalize for transfection efficiency, the plasmids for EBV protein expression library, together with cGAS and STING expression plasmids. Cells were harvested 24 hours post-transfection for dual-luciferase assay. The mean values of three biological replicates (n = 3) were represented by the bars and their respective standard deviations were depicted as the error bars. (B-D) BPLF1 inhibits cGAS-STING-, RIG-I- and TBK1-induced IFNβ-promoter activation. HEK293 cells were transfected with IFNB-Luc, TK-Luc, and increasing doses of BPLF1 expression plasmid. IFNB-Luc reporter expression was stimulated with expression plasmids for cGAS + STING (B), RIG-I N (C) and TBK1 (D). Cells were harvested 24 hours post-transfection for dual-luciferase assay. The mean values of three biological replicates (n = 3) were represented by the bars and their respective standard deviations were depicted as the error bars. The statistical significance for the difference between the indicated groups was analyzed using two-tailed Student’s t-test for paired samples and the ranges of the p values were indicated (*: p < 0.05; **: p < 0.01; and ***: p < 0.001).

Article Snippet: Rabbit anti-cGAS (D1D3G), rabbit anti-STING (D2P2F) and rabbit anti-TBK1 (D1B4) antibodies were from Cell Signaling Technology.

Techniques: Functional Assay, Transfection, Luciferase, Plasmid Preparation, Expressing, Activation Assay, Two Tailed Test

Journal: PLOS Pathogens

Article Title: Suppression of cGAS- and RIG-I-mediated innate immune signaling by Epstein-Barr virus deubiquitinase BPLF1

doi: 10.1371/journal.ppat.1011186

Figure Lengend Snippet: (A-D) Specific inhibition of cGAS-STING activity by BPLF1. HEK293 cells were transfected with IFNB-Luc, TK-Luc, either one or two doses of BPLF1 expression plasmid, and expression plasmids for cGAS + STING (A), IRF3 (B) or IRF3-5D (C). Alternatively, a Firefly luciferase reporter driven by the SV40-promoter (SV40-Luc) and TK-Luc promoter were co-transfected with BPLF1 expression plasmid into HEK293 cells (D). Cells were harvested 24 hours post-transfection for dual-luciferase assay. (E) A schematic diagram depicting the targeting regions of the two gRNAs (guide-1 and guide-2) on the EBV genome was presented together with dCas9, four tandem copies of HSV-1 VP16 molecules (VP64) and the activator helper complex. (F) The cDNA sequences of the two gRNAs. (G) Induction of BPLF1 expression by CRISPR-a. HEK293M81 cells were transfected with lentiMPHv2 and lentiSAMv2 carrying guide-1 and guide-2. The total cellular RNA was harvested 48 hours post-transfection and the BPLF1 mRNA was measured by RT-qPCR. The BPLF1 mRNA expression levels were normalized to those of endogenous GAPDH transcript. (H) IFN antagonism of BPLF1 induced to express from EBV genome by CRISPR-a. HEK293M81 cells were transfected with IFNB-Luc, TK-Luc, guide-1 or guide-2 plasmids and expression plasmids for cGAS + STING, RIG-I N and TBK1. (I) Dose-dependent IFN antagonism of BPLF1 expressed from EBV genome. HEK293M81 cells were transfected with IFNB-Luc, TK-Luc, increasing doses of guide-2 plasmid, and TBK1 expression plasmid for dual luciferase assays. (J-L) BPLF1 inhibits cGAS-STING-, RIG-I- and TBK1-induced activation of IRF3-binding elements. HEK293 cells were transfected with a Firefly luciferase reporter plasmid driven by IRF3-Luc, TK-Luc, increasing doses of BPLF1 expression plasmid, and expression plasmids for cGAS + STING (J), RIG-I N (K) or TBK1 (L). IκB super-repressor was also over-expressed in (J). Cells were harvested 24 hours post-transfection for dual-luciferase assay. The mean values of three biological replicates (n = 3) were represented by the bars and their respective standard deviations were depicted as the error bars. The statistical significance among selected samples was analyzed using two-tailed Student’s t-test for paired samples and the ranges of the p values were indicated (*: p < 0.05; **: p < 0.01; and n.s.: not significant or p > 0.05).

Article Snippet: Rabbit anti-cGAS (D1D3G), rabbit anti-STING (D2P2F) and rabbit anti-TBK1 (D1B4) antibodies were from Cell Signaling Technology.

Techniques: Inhibition, Activity Assay, Transfection, Expressing, Plasmid Preparation, Luciferase, CRISPR, Quantitative RT-PCR, Activation Assay, Binding Assay, Two Tailed Test

Journal: PLOS Pathogens

Article Title: Suppression of cGAS- and RIG-I-mediated innate immune signaling by Epstein-Barr virus deubiquitinase BPLF1

doi: 10.1371/journal.ppat.1011186

Figure Lengend Snippet: (A) Expression of BPLF1 C61A mutant. BPLF1 WT and C61A were transiently expressed in HEK293 cells. Endogenous α-tubulin was used for normalization. (B-D) IRF3 activation by C61A mutant. The indicated combinations of BPLF1 expression plasmid, mutant C61A expression construct as well as IRF3-Luc and TK-Luc reporter plasmids were transfected into HEK293 cells together with cGAS + STING (B), RIG-I N (C) or TBK1 (D) expression plasmids. Cells were harvested 24 hours post-transfection for dual-luciferase assay. (E) Effect of C61A on IFN-β induction. HEK293 cells were transfected with cGAS, STING, BPLF1 and mutant C61A expression plasmids. The total cellular RNA was harvested 48 hours post-transfection and the IFN-β transcripts expression levels were measured by RT-qPCR. The IFNβ transcript expression levels were normalized to those of endogenous GAPDH transcript. The mean values of three biological replicates (n = 3) were represented by the bars and their respective standard deviations were depicted as the error bars. (F, G) The DUB activity of BPLF1 promotes EBV infection. BPLF1 and C61A expression plasmids were transfected into HEK293 cells together with cGAS + STING expression plasmids (panels 1–3) and TBK1 expression plasmid (panels 4–6). After 24 hours post- transfection, cells were infected with 1 m.o.i. of freshly prepared EBV M81. Microscopic images were captured (F) and GFP signals were analyzed by flow cytometry 48 hours post-infection (G). The percentages of GFP + cells were normalized to the mock infection group. The mean values of three biological replicates (n = 3) were represented by the bars and their respective standard deviations were depicted as the error bars. The statistical significance among selected samples was analyzed using two-tailed Student’s t-test for paired samples and the ranges of the p values were indicated (*: p < 0.05; and **: p < 0.01; ***: p < 0.001).

Article Snippet: Rabbit anti-cGAS (D1D3G), rabbit anti-STING (D2P2F) and rabbit anti-TBK1 (D1B4) antibodies were from Cell Signaling Technology.

Techniques: Expressing, Mutagenesis, Activation Assay, Plasmid Preparation, Construct, Transfection, Luciferase, Quantitative RT-PCR, Activity Assay, Infection, Flow Cytometry, Two Tailed Test

Journal: PLOS Pathogens

Article Title: Suppression of cGAS- and RIG-I-mediated innate immune signaling by Epstein-Barr virus deubiquitinase BPLF1

doi: 10.1371/journal.ppat.1011186

Figure Lengend Snippet: (A) Influence of BPLF1 and C61A on TBK1 ubiquitination. BPLF1, C61A and TBK1 expression plasmids were transfected into HEK293T cells together with HA-Ub-WT and lysine-free ubiquitin (HA-Ub-K0). (B) Influence of BPLF1 and C61A on TBK1 ubiquitination of different types. BPLF1, C61A and TBK1 expression plasmids were transfected into HEK293T cells together with HA-Ub-WT and HA-K63-Ub and HA-K48-Ub. Cells were harvested 24 hours after transfection for co-immunoprecipitation. The Flag-tagged TBK1 molecules were pulled down by anti-Flag antibodies. The bound fraction of the immunoprecipitates (IP) and the total lysates (input) were analyzed by Western blotting (WB) with anti-HA, anti- Flag and anti-β-tubulin antibodies. Long exposure was conducted for visualizing proteins in the upper panel and short exposure was conducted for visualizing proteins in the lower panel.

Article Snippet: Rabbit anti-cGAS (D1D3G), rabbit anti-STING (D2P2F) and rabbit anti-TBK1 (D1B4) antibodies were from Cell Signaling Technology.

Techniques: Expressing, Transfection, Immunoprecipitation, Western Blot

Journal: PLOS Pathogens

Article Title: Suppression of cGAS- and RIG-I-mediated innate immune signaling by Epstein-Barr virus deubiquitinase BPLF1

doi: 10.1371/journal.ppat.1011186

Figure Lengend Snippet: (A, B) Co-immunoprecipitation of BPLF1 and STING. Myc-BPLF1 and Flag-STING expression plasmids were expressed in HEK293T cells. (C) No association between BPLF1 and TBK1. Myc-BPLF1 and Flag-TBK1 were expressed in HEK293T cells. Cells were harvested 24 hours after transfection for co-immunoprecipitation. The Flag-tagged STING and TBK1 proteins were pulled down by anti-Flag antibodies. The bound fraction of the immunoprecipitates (IP) and the total lysates (input) were analyzed by Western blotting (WB) with anti-Myc, anti-Flag and anti-α-tubulin antibodies.

Article Snippet: Rabbit anti-cGAS (D1D3G), rabbit anti-STING (D2P2F) and rabbit anti-TBK1 (D1B4) antibodies were from Cell Signaling Technology.

Techniques: Immunoprecipitation, Expressing, Transfection, Western Blot

Journal: PLOS Pathogens

Article Title: Suppression of cGAS- and RIG-I-mediated innate immune signaling by Epstein-Barr virus deubiquitinase BPLF1

doi: 10.1371/journal.ppat.1011186

Figure Lengend Snippet: IRF3 dimerization assay by native PAGE. BPLF1 and mutant C61A were transiently overexpressed in HEK293 cells together with TBK1 and V5-IRF3. Cells were harvested 48 hours after transfection. The cell lysates were subjected to SDS-PAGE and native PAGE. The lysates were analyzed by Western blotting by anti-V5, anti-Flag, anti-TBK1 and anti-α-tubulin antibodies.

Article Snippet: Rabbit anti-cGAS (D1D3G), rabbit anti-STING (D2P2F) and rabbit anti-TBK1 (D1B4) antibodies were from Cell Signaling Technology.

Techniques: Clear Native PAGE, Mutagenesis, Transfection, SDS Page, Western Blot

Journal: PLOS Pathogens

Article Title: Suppression of cGAS- and RIG-I-mediated innate immune signaling by Epstein-Barr virus deubiquitinase BPLF1

doi: 10.1371/journal.ppat.1011186

Figure Lengend Snippet: (A) BPLF1 mRNA expression in EBV + cells. HEK293, NP460EBV, HEK293p2089#1–2, HEK293p2089-C61A#1–4 cells were seeded into 6-well plates. The total cellular RNA was harvest 48 hours after transfection and the BPLF1 transcript expression levels were determined by RT-qPCR and the relative BPLF1 transcript expression levels were normalized to endogenous GAPDH transcript levels. (B) STING ubiquitination in cells carrying BPLF1-inactive EBV. HEK293, HEK293p2089 and HEK293p2089-C61A#1–4 cells were seeded into 60mm dishes. Cells were harvested 48 hours after transfection for co-immunoprecipitation. The Flag-tagged STING molecules were pulled down by the anti-Flag antibodies. The bound fraction of the immunoprecipitates (IP) and the total lysates (input) were analyzed by Western blotting (WB) with anti-HA, anti-Flag and anti-β-tubulin antibodies. (C) IFNB-Luc activation in cells carrying BPLF1-inactive EBV. IFNB-Luc and TK-Luc reporter plasmids were transfected into HEK293, HEK293p2089#1–2, HEK293p2089-C61A#1–4 cells together with cGAS and STING expression plasmids. (D) IFN-β induction in cells carrying BPLF1-inactive EBV. Cells were harvested 24 hours post-transfection for dual-luciferase assay. HEK293, HEK293p2089#1–2, HEK293p2089-C61A#1–4 cells were transfected with cGAS and STING expression plasmids. The total cellular RNA was harvested 48 hours post-transfection and the IFNβ transcripts expression levels were measured by RT-qPCR. The IFN-β transcript expression levels were normalized by the endogenous GAPDH transcripts level. The mean values of three biological replicates (n = 3) were represented by the bars and their respective standard deviations were depicted as the error bars. The statistical significance among selected samples were analyzed using two-tailed Student’s t-test for paired samples and the p- values were indicated. (E) IFNB-Luc activation in cells stably carrying BPLF1-inactive EBV. WT p2089 and BPLF1C61A p2089 EBV BAC were transfected into HEK293 cells for stable cell construction. The transfected cells were selected with hygromycin for about a week and the survival cells were then subject for IFN-β analysis. The stable cells were transfected with TBK1 or cGAS and STING for IFN-β production. TPA is added at 24 hours post-transfection to stimulate the lytic transcript production. Cells were harvested in the next day and RT-qPCR were performed to measure the IFN-β transcript level. GAPDH was used for normalization.

Article Snippet: Rabbit anti-cGAS (D1D3G), rabbit anti-STING (D2P2F) and rabbit anti-TBK1 (D1B4) antibodies were from Cell Signaling Technology.

Techniques: Expressing, Transfection, Quantitative RT-PCR, Immunoprecipitation, Western Blot, Activation Assay, Luciferase, Two Tailed Test, Stable Transfection

Journal: PLOS Pathogens

Article Title: Antagonism of ALAS1 by the Measles Virus V protein contributes to degradation of the mitochondrial network and promotes interferon response

doi: 10.1371/journal.ppat.1011170

Figure Lengend Snippet: A) Western blot analysis of TBK1, TBK1-P, IRF3, IRF3-P, RIG-I, MAD5, MAVS, STING, IKK ε and cGAS at 24 hpi by MeV at MOI 1 and 3 and compared to non-infected cells (NI) and cells infected by heat-inactivated virus (HI). β-Actin protein expression was used as loading control. kDa: kilo Dalton. B-D) Gene profiling of the IFN pathway after infection by MeV at 24 hpi: B) RIG-I , C) MDA-5 , D) RNA polymerase III, expressions were normalized to RPL13A housekeeping gene expression level. Mean values and s.e.m. were calculated for three independent experiments in duplicate (n = 6). E) A3A relative expression, normalized to RPL13A housekeeping gene expression upon MeV infection at 0.1, 1 and 3 MOI and concomitant treatment with ML-60218 (inhibitor of RNA polymerase III) at 25 μM or 50 μM. Unpaired two-side Student’s t-test, **, p < 0.05, ***, p < 0.005 and ns: not statistically significant.

Article Snippet: MDA5 (D74E4, rabbit mAb #5321), STING (antibody #3337), phospho-IRF-3 (Ser396) (4D4G, rabbit mAb #4947), IRF-3 (D6I4C, XP rabbit mAb #11904), RIG-I (D14G6, rabbit mAb #3743), MAVS (antibody #3993), TBK1/NAK (D1B4, rabbit mAb #3504), phospho-TBK1/NAK (Ser 172) (D52C2, rabbit mAb, #5483), IKKε (D20G4, rabbit mAb, #2905), cGAS (E9G9G, rabbit mAb, #83623), beta actin HRP conjugate (13E5, rabbit mAb # 5125), anti-rabbit IgG HRP-linked (antibody #7074), histone H2AX (antibody #2595), antibodies were from Cell Signaling Technology.

Techniques: Western Blot, Infection, Expressing

Journal: PLOS Pathogens

Article Title: Antagonism of ALAS1 by the Measles Virus V protein contributes to degradation of the mitochondrial network and promotes interferon response

doi: 10.1371/journal.ppat.1011170

Figure Lengend Snippet: A) Western blot analysis of TBK1, TBK1-P, IRF3, IRF3-P, RIG-I, MAD5, MAVS, STING, IKK ε and cGAS at 24 hpi by MeV at MOI 1 and 3 and compared to non-infected cells (NI) and cells infected by heat-inactivated virus (HI). β-Actin protein expression was used as loading control. kDa: kilo Dalton. B-D) Gene profiling of the IFN pathway after infection by MeV at 24 hpi: B) RIG-I , C) MDA-5 , D) RNA polymerase III, expressions were normalized to RPL13A housekeeping gene expression level. Mean values and s.e.m. were calculated for three independent experiments in duplicate (n = 6). E) A3A relative expression, normalized to RPL13A housekeeping gene expression upon MeV infection at 0.1, 1 and 3 MOI and concomitant treatment with ML-60218 (inhibitor of RNA polymerase III) at 25 μM or 50 μM. Unpaired two-side Student’s t-test, **, p < 0.05, ***, p < 0.005 and ns: not statistically significant.

Article Snippet: MDA5 (D74E4, rabbit mAb #5321), STING (antibody #3337), phospho-IRF-3 (Ser396) (4D4G, rabbit mAb #4947), IRF-3 (D6I4C, XP rabbit mAb #11904), RIG-I (D14G6, rabbit mAb #3743), MAVS (antibody #3993), TBK1/NAK (D1B4, rabbit mAb #3504), phospho-TBK1/NAK (Ser 172) (D52C2, rabbit mAb, #5483), IKKε (D20G4, rabbit mAb, #2905), cGAS (E9G9G, rabbit mAb, #83623), beta actin HRP conjugate (13E5, rabbit mAb # 5125), anti-rabbit IgG HRP-linked (antibody #7074), histone H2AX (antibody #2595), antibodies were from Cell Signaling Technology.

Techniques: Western Blot, Infection, Expressing

Journal: Emerging Microbes & Infections

Article Title: NSUN2-mediated M 5 c methylation of IRF3 mRNA negatively regulates type I interferon responses during various viral infections

doi: 10.1080/22221751.2023.2178238

Figure Lengend Snippet: NSUN2 inhibits the expression level of IRF3. (a) Dual-luciferase assay analysing a luciferase reporter plasmid for the IRF3-responsive promoter containing positive regulatory domains III and I of the IFN-β promoter (PRDIII-I-Luc) in HEK293T cells in 24-well plates transfected for 36 h with the RIG-N, MDA5-N, MAVS, TBK1, and IRF3-5D expression plasmids, as indicated, with co-transfection with empty vector or NSUN2. (b) Dual-luciferase analysis of PRDIII-I-Luc in HEK293T cells in 24-well plates transfected for 36 h with the indicated RIG-N, MDA5-N, MAVS, TBK1, and IRF3-5D expression plasmids with co-transfection with siControl or siNSUN2-1. (c) Immunoblot analysis in HEK293T cells transfected with vector or NSUN2 for 36 h, with or without infection by SeV for another 12 h. (d) Immunoblot analysis in wild-type HEK293T cells or NSUN2 −/− HEK293T cells with or without infection by SeV for 12 h. (e) Immunoblot analysis in wild-type A549 cells or NSUN2 −/− A549 cells with infection by SeV for 0, 4, 8, and 12 h. (f) Immunoblot analysis in wild-type HEK293T cells or NSUN2 −/− HEK293T cells, with infection by SeV for 0, 4, 8, and 12 h. (g) qPCR analysis of IFNB1 mRNA in wild-type HEK293T cells or NSUN2 −/− HEK293T cells transfected with vector or NSUN2, with co-transfection with vector or IRF3-FL, as indicated, with infection by SeV for 12 h. (h) qPCR analysis of VSV-G or HSV-1-UL-30 RNA in Irf3 −/− Irf7 −/− MEFs transfected with vector or NSUN2, with co-transfection with vector or IRF3-FL, as indicated, with infection by VSV or HSV-1 for 12 h. Data are representative of three independent experiments and were analysed by two-tailed unpaired t test (or by two-factor ANOVA test for 2 h and 2i). Graphs show the mean ± SD (n = 3) derived from three independent experiments. NS, not significant for P > 0.05, * P < 0.05, ** P < 0.01, *** P -< 0.001.

Article Snippet: The antibodies used were as follows: rabbit anti-NSUN2 (Proteintech, 20854-1-AP), rabbit anti-Phospho-IRF-3-Ser396 (CST, 83611S), rabbit anti-IRF3 (Proteintech, 11312-1-AP), rabbit anti-phospho-TBK1/NAK-Ser172 (CST, 14590S), rabbit anti-TBK1/NAK (CST, 38066S), mouse anti-HA (Sigma, H6908), rabbit anti-HA (Sigma, H3663), mouse anti-Flag (Proteintech, 66008-3-Ig), rabbit anti-Flag (Sigma, SAB4301135), mouse anti-m 5 C antibody (Abcam, ab10805), mouse anti-GAPDH (Proteintech, 60004-1-Ig), mouse anti-β-actin (Proteintech, 66009-1-Ig).

Techniques: Expressing, Luciferase, Plasmid Preparation, Transfection, Cotransfection, Western Blot, Infection, Two Tailed Test, Derivative Assay

Journal: Emerging Microbes & Infections

Article Title: NSUN2-mediated M 5 c methylation of IRF3 mRNA negatively regulates type I interferon responses during various viral infections

doi: 10.1080/22221751.2023.2178238

Figure Lengend Snippet: NSUN2 interacts with IRF3 mRNA and induces its degradation. (a) Coimmunoprecipitation (IP) and immunoblot (IB) analysis of HEK293T cells transfected with plasmids encoding HA-NSUN2 and Flag-IRF3. (b) Immunoprecipitation by HA-Tag-conjugated beads and immunoblot analysis of HEK293T cells transfected with plasmids encoding HA-NSUN2, with SeV infection for 8 h, followed by RNA extraction and qPCR analysis of combined IRF3 mRNA. (c) qPCR analysis of IRF3 mRNA and TBK1 mRNA in HEK293T cells transfected with siControl or siRNAs targeting NSUN2, with or without infection by SeV for 8 h. (d) qPCR analysis of IRF3 mRNA and TBK1 mRNA in wild-type HEK293T cells or NSUN2 −/− HEK293T cells, with or without infection by SeV for 8 h. (e) Stability analysis of IRF3 mRNA and TBK1 mRNA in wild-type HEK293T cells or NSUN2 −/− HEK293T cells with treatment of actinomycin D (ActD) for 0, 6, 12, and 18 h. (f) The piled reads of IRF3 mRNA from NSUN2-LACE-seq from SeV-infected cells: NSUN2 −/− HEK293T (lower) and NSUN2 −/− HEK293T with reconstitution of exogenous HA-NSUN2 (upper). Y-axis represents the normalized signals along the gene. Data are representative of three independent experiments and were analysed by two-tailed unpaired t test. Graphs show the mean ± SD (n = 3) derived from three independent experiments (or two independent experiments for 3f). NS, not significant for P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The antibodies used were as follows: rabbit anti-NSUN2 (Proteintech, 20854-1-AP), rabbit anti-Phospho-IRF-3-Ser396 (CST, 83611S), rabbit anti-IRF3 (Proteintech, 11312-1-AP), rabbit anti-phospho-TBK1/NAK-Ser172 (CST, 14590S), rabbit anti-TBK1/NAK (CST, 38066S), mouse anti-HA (Sigma, H6908), rabbit anti-HA (Sigma, H3663), mouse anti-Flag (Proteintech, 66008-3-Ig), rabbit anti-Flag (Sigma, SAB4301135), mouse anti-m 5 C antibody (Abcam, ab10805), mouse anti-GAPDH (Proteintech, 60004-1-Ig), mouse anti-β-actin (Proteintech, 66009-1-Ig).

Techniques: Western Blot, Transfection, Immunoprecipitation, Infection, RNA Extraction, Two Tailed Test, Derivative Assay

Journal: Emerging Microbes & Infections

Article Title: NSUN2-mediated M 5 c methylation of IRF3 mRNA negatively regulates type I interferon responses during various viral infections

doi: 10.1080/22221751.2023.2178238

Figure Lengend Snippet: NSUN2 catalyzes the formation of m 5 C methylation of IRF3 mRNA both exogenously and endogenously. (a) Schematic diagram of the IRF3 mRNA segments used for in vitro methylation assays and bisulfite RNA sequencing. (b) In vitro m 5 C methylation assays using recombinant GST-NSUN2 and the in vitro transcripts. (c) In vitro m 5 C methylation assays using recombinant GST-NSUN2 and the in vitro transcribed segments of IRF3 mRNA depicted in (a). (d) m 5 C dot blot analysis of endogenous IRF3 mRNA (200 ng) pulled down by IRF3 CHIRP probes in wild-type HEK293T cells or NSUN2 −/− HEK293T cells with or without exogenous NSUN2 overexpression. Equal IRF3 mRNAs were also loaded and verified by methylene blue (MB) staining. (e) The m 5 C-RIP-qPCR analysis of the m 5 C methylated IRF3 mRNA immunoprecipitated by m 5 C antibody from wild-type HEK293T cells or NSUN2 −/− HEK293T cells, with or without exogenous NSUN2 expression. TBK1 was used as a negative control. (f-g) RNA-seq of HEK293T cells or HEK293T cells with NSUN2 overexpression, with infection by SeV. The heatmap (f) shows the expression levels of ISGs and several signalling molecules. The genes that we focus on are labelled with asterisks. The column diagram (g) shows the SeV replication levels. (h) In vitro m 5 C methylation assays using recombinant GST-NSUN2 and different mutant proteins.

Article Snippet: The antibodies used were as follows: rabbit anti-NSUN2 (Proteintech, 20854-1-AP), rabbit anti-Phospho-IRF-3-Ser396 (CST, 83611S), rabbit anti-IRF3 (Proteintech, 11312-1-AP), rabbit anti-phospho-TBK1/NAK-Ser172 (CST, 14590S), rabbit anti-TBK1/NAK (CST, 38066S), mouse anti-HA (Sigma, H6908), rabbit anti-HA (Sigma, H3663), mouse anti-Flag (Proteintech, 66008-3-Ig), rabbit anti-Flag (Sigma, SAB4301135), mouse anti-m 5 C antibody (Abcam, ab10805), mouse anti-GAPDH (Proteintech, 60004-1-Ig), mouse anti-β-actin (Proteintech, 66009-1-Ig).

Techniques: Methylation, In Vitro, RNA Sequencing Assay, Recombinant, Dot Blot, Over Expression, Staining, Immunoprecipitation, Expressing, Negative Control, Infection, Mutagenesis