Journal: Frontiers in Immunology

Article Title: Identification of interferon-stimulated genes with modulated expression during hepatitis E virus infection in pig liver tissues and human HepaRG cells

doi: 10.3389/fimmu.2023.1291186

Figure Lengend Snippet: List of primers used for the quantification of human gene expression by RT-qPCR.

Article Snippet: The membrane was then incubated with the required dilution of specific antibodies raised against ORF2 (mouse, 1/2000 dilution, clone 1E6, Merck Millipore), actin (mouse, 1/2000 dilution, clone M2, Sigma), DDX58 (mouse, 1/1000 dilution, clone Alme-1, AdipoGen Life Sciences), IFIH1 (rabbit, 1/1000 dilution, AT113, ALX-210-935, Enzo Life Sciences), or IRF1 (rabbit, 1/1000 dilution, VPA00801, Bio-Rad).

Techniques: Gene Expression

Journal: Frontiers in Immunology

Article Title: Identification of interferon-stimulated genes with modulated expression during hepatitis E virus infection in pig liver tissues and human HepaRG cells

doi: 10.3389/fimmu.2023.1291186

Figure Lengend Snippet: Validation of the PCR array data. (A) HepaRG cells were infected or not with HEV-3f for 100 days at MOI 100 and the expression of selected genes shown by PCR array to be up-regulated or unchanged was analyzed by RT-qPCR. The results shown are the geometric means ± SD from 3 to 4 replicate samples and are representative of 2 independent experiments. GUSB , B2M and GAPDH were used as reference genes. Unpaired t-test with Welch’s correction Mann-Whitney test, *: p<0.05 (B) Immunoblot showing the expression of the protein encoded by IFIH1 , DDX58 and IRF1 in HepaRG cells infected with HEV-3f or not (NI) for 100 days at MOI 100 for 3 replicate samples. Representative blots from 2 independent experiments are shown. ORF2 and actin protein levels were also detected as control of infection and loading, respectively. (C) Detection of CXCL10 in the supernatant of non-infected HepaRG (NI) or HepaRG cells infected with HEV-3f for 100 days at MOI 100 (GE/cell) by ELISA. The results shown are the means ± SD from 4 replicate samples and are representative of 2 independent experiments. Unpaired t-test with Welch’s correction, *: p<0.05.

Article Snippet: The membrane was then incubated with the required dilution of specific antibodies raised against ORF2 (mouse, 1/2000 dilution, clone 1E6, Merck Millipore), actin (mouse, 1/2000 dilution, clone M2, Sigma), DDX58 (mouse, 1/1000 dilution, clone Alme-1, AdipoGen Life Sciences), IFIH1 (rabbit, 1/1000 dilution, AT113, ALX-210-935, Enzo Life Sciences), or IRF1 (rabbit, 1/1000 dilution, VPA00801, Bio-Rad).

Techniques: Biomarker Discovery, Infection, Expressing, Quantitative RT-PCR, MANN-WHITNEY, Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: Uncovering the Interaction between TRAF1 and MAVS in the RIG-I Pathway to Enhance the Upregulation of IRF1/ISG15 during Classical Swine Fever Virus Infection

doi: 10.3390/cells13131165

Figure Lengend Snippet: Primers used in this study.

Article Snippet: Anti-IRF1 (11335-1-AP), anti-RIG-I (20566-1-AP), anti-Myc tag (16286-1-AP), and anti-Flag tag (20543-1-AP) antibodies were purchased from Proteintech™ Co., Ltd. (Wuhan, China).

Techniques: Sequencing, Knockdown

Journal: Cells

Article Title: Uncovering the Interaction between TRAF1 and MAVS in the RIG-I Pathway to Enhance the Upregulation of IRF1/ISG15 during Classical Swine Fever Virus Infection

doi: 10.3390/cells13131165

Figure Lengend Snippet: TRAF1 expression positively correlated with the RIG-I/MAVS pathway during CSFV infection. ( A – F ) The CSFV gRNA and the mRNA levels of TRAF1, RIG-I, MAVS, IRF1, and ISG15 proteins were detected in PK-15 cells infected with the CSFV Shimen strain (MOI = 1) at 3, 6, 9, 12, 24, 36, 48, and 72 hpi by RT-qPCR analysis. ( G ) The protein levels of CSFV E2, TRAF1, RIG-I, MAVS, IRF1, and ISG15 were detected in PK-15 cells infected with the CSFV Shimen strain (MOI = 1) at 3, 6, 9, 12, 24, 36, 48, and 72 hpi by WB analysis. ( H – M ). ( N ) Pearson analysis of the CSFV E2, TRAF1, RIG-I, MAVS, IRF1, and ISG15 protein bands. The gray value of each target band from WB was measured by comparing them to β-actin using ImageJ software version 1.8.0. All statistical analysis results were derived from comparisons between the experimental group and the Mock group (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Anti-IRF1 (11335-1-AP), anti-RIG-I (20566-1-AP), anti-Myc tag (16286-1-AP), and anti-Flag tag (20543-1-AP) antibodies were purchased from Proteintech™ Co., Ltd. (Wuhan, China).

Techniques: Expressing, Infection, Quantitative RT-PCR, Software, Derivative Assay

Journal: Cells

Article Title: Uncovering the Interaction between TRAF1 and MAVS in the RIG-I Pathway to Enhance the Upregulation of IRF1/ISG15 during Classical Swine Fever Virus Infection

doi: 10.3390/cells13131165

Figure Lengend Snippet: Fold-changes of the levels of CSFV E2, TRAF1, RIG-I, MAVS, and ISG15 proteins in PK-15 cells following CSFV infection.

Article Snippet: Anti-IRF1 (11335-1-AP), anti-RIG-I (20566-1-AP), anti-Myc tag (16286-1-AP), and anti-Flag tag (20543-1-AP) antibodies were purchased from Proteintech™ Co., Ltd. (Wuhan, China).

Techniques: Infection

Journal: Cells

Article Title: Uncovering the Interaction between TRAF1 and MAVS in the RIG-I Pathway to Enhance the Upregulation of IRF1/ISG15 during Classical Swine Fever Virus Infection

doi: 10.3390/cells13131165

Figure Lengend Snippet: TRAF1 downregulation by siRNA-enhanced CSFV replication. ( A – F ) The CSFV gRNA and the mRNA levels of TRAF1, RIG-I, MAVS, IRF1, and ISG15 proteins were detected in different kinds of PK-15 cells, such as infection of CSFV Shimen strain at an MOI of 1.0 as a positive control, temporary transfection of 100 nM TRAF1 siRNA 6 h then infection of CSFV Shimen strain, temporary transfection of 100 nM control siRNA 6 h then infection of CSFV Shimen strain as a negative control by RT-qPCR analysis at 24, 36, and 48 hpi. ( G ) The experimental treatment was consistent with the ( A ) and WB analysis at 24, 36, and 48 hpi. ( H – M ) The gray value of each target band from WB was measured by comparing them to β-actin using ImageJ software version 1.8.0. ( N ) The experimental treatment was consistent with ( A ). Culture supernatant from PK-15 cells subjected to different treatments was collected, and the viral titer was tested by IFA at 12, 24, 36, 48, and 60 hpi. All statistical analysis results were derived from comparisons between the experimental group and the Mock group (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Anti-IRF1 (11335-1-AP), anti-RIG-I (20566-1-AP), anti-Myc tag (16286-1-AP), and anti-Flag tag (20543-1-AP) antibodies were purchased from Proteintech™ Co., Ltd. (Wuhan, China).

Techniques: Infection, Positive Control, Transfection, Control, Negative Control, Quantitative RT-PCR, Software, Derivative Assay

Journal: Cells

Article Title: Uncovering the Interaction between TRAF1 and MAVS in the RIG-I Pathway to Enhance the Upregulation of IRF1/ISG15 during Classical Swine Fever Virus Infection

doi: 10.3390/cells13131165

Figure Lengend Snippet: TRAF1 overexpression inhibited CSFV replication by positive regulation of IRF1 and ISG15. ( A – F ) The CSFV gRNA and mRNA levels of TRAF1, RIG-I, MAVS, IRF1, and ISG15 proteins were detected in different kinds of PK-15 cells, such as infection of CSFV Shimen strain at an MOI of 1.0 as a positive control, temporary transfection of 1.0 µg pcDNA3.0-TRAF1 -Flag vector 12 h then infection of CSFV Shimen strain, temporary transfection of 1.0 µg empty vector 12 h then infection of CSFV Shimen strain as negative control by RT-qPCR analysis at 24, 36, and 48 hpi. ( G ) The experimental treatment was consistent with ( A ) and the WB analysis at 24, 36, and 48 hpi. ( H – M ) The gray value of each target band from WB was measured by comparing them to β-actin using ImageJ software version 1.8.0. ( N ) The experimental treatment was consistent with ( A ). Culture supernatant from PK-15 cells subjected to different treatments was collected, and the viral titer was tested by IFA at 12, 24, 36, 48, and 60 hpi. All statistical analysis results were derived from comparisons between the experimental group and the Mock group (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Anti-IRF1 (11335-1-AP), anti-RIG-I (20566-1-AP), anti-Myc tag (16286-1-AP), and anti-Flag tag (20543-1-AP) antibodies were purchased from Proteintech™ Co., Ltd. (Wuhan, China).

Techniques: Over Expression, Infection, Positive Control, Transfection, Plasmid Preparation, Negative Control, Quantitative RT-PCR, Software, Derivative Assay

Journal: Cells

Article Title: Uncovering the Interaction between TRAF1 and MAVS in the RIG-I Pathway to Enhance the Upregulation of IRF1/ISG15 during Classical Swine Fever Virus Infection

doi: 10.3390/cells13131165

Figure Lengend Snippet: TRAF1 participated in the RIG-I/MAVS pathway against CSFV infection. RIG-I identifies short plus-stranded RNA of CSFV. After activation of RIG-I, its CARD region merges with the CARD region of MAVS to activate downstream signal molecules. TRAF1 binds to the PRR domain of MAVS to promote IRF1 activation and subsequent stimulation of ISG15 expression.

Article Snippet: Anti-IRF1 (11335-1-AP), anti-RIG-I (20566-1-AP), anti-Myc tag (16286-1-AP), and anti-Flag tag (20543-1-AP) antibodies were purchased from Proteintech™ Co., Ltd. (Wuhan, China).

Techniques: Infection, Activation Assay, Expressing

Journal: Cell Death and Differentiation

Article Title: Lactylation of NAT10 promotes N 4 ‐acetylcytidine modification on tRNA Ser-CGA-1-1 to boost oncogenic DNA virus KSHV reactivation

doi: 10.1038/s41418-024-01327-0

Figure Lengend Snippet: A The total RNAs from iSLK-KSHV NAT10 +/- cells (NAT10 +/- ) and iSLK-KSHV NAT10 +/+ cells (WT) were subjected to dot blot assay with anti-ac 4 C antibody. Methylene blue staining was used as the internal control. B The heatmap for differentially expressed tRNAs in cells shown as in ( A ) by tRNA acRIP-seq analysis. The pseudo-color represented as the fold enrichment (IP/Input) of WT and NAT10 +/− group, respectively. Any tRNAs not detected in tRNA acRIP-seq were plotted as white in the heatmap. C The total RNAs from cells shown as in ( A ) were subjected to Northern blot with probes to tRNA Ser-CGA-1-1 , tRNA Ser-CGA-4-1 , tRNA Leu-TAG-3-1 , tRNA Ser-AGA , tRNA Leu-TAA , and U6 as the control. D The RT-qPCR analysis for the ribosome-nascent chain-complex-bound mRNA (RNC-qPCR) of representative viral genes of KSHV (RTA, K5, K8, ORF45, ORF57, vIRF1, vIL-6, vBCL-2, ORF65, and K8.1) in cells shown as in ( A ). *** P < 0.001 by Student’s t -test. E Schematic representation of the acetylation site mutation of tRNA Ser-CGA-1-1 (Upper) and tRNA Leu-TAG-3-1 (Lower). The C (marked in red) at position 12 was mutated to U (marked in dark blue). F The total RNAs from iSLK-KSHV cells with overexpression of the wild type tRNA Ser-CGA-1-1 or tRNA Leu-TAG-3-1 (WT), mutant tRNA Ser-CGA-1-1 or tRNA Leu-TAG-3-1 (Mut), and their control empty vector ( EV ) for 48 h were respectively subjected to Northern blot with probes to tRNA Ser-CGA-1-1 , tRNA Leu-TAG-3-1 and U6 as the control. G . The RT-qPCR analysis for the ribosome-nascent chain-complex-bound mRNA (RNC-qPCR) of representative viral genes of KSHV (RTA, K5, K8, vIRF1, vIL-6, vBCL-2, and ORF65) in iSLK-KSHV cells with overexpression of the wild type (tRNA Ser-CGA-1-1 -WT) or its mutant (tRNA Ser-CGA-1-1 -Mut) tRNA Ser-CGA-1-1 for 48 h, and their control (tRNA-EV). *** P < 0.001 by Student’s t -test. H The expression levels of vIRF1 and NAT10 in iSLK-KSHV cells with overexpression of the wild type (WT), mutant (Mut) tRNA Ser-CGA-1-1 , and their control (EV) for 48 h were detected by Western blot. I The SDS gel analysis of the in vitro translation reaction supplemented with the wild type (tRNA Ser-CGA-1-1 -WT) or the mutant (tRNA Ser-CGA-1-1 -Mut) tRNA Ser-CGA-1-1 , in comparison with reaction mixtures that not containing mRNA (No mRNA). J By the assessment of ORF26, real-time DNA-PCR for cells shown as in ( H ) was performed to detect viral copy number after doxycycline stimulation for 72 h. *** P < 0.001 by Student’s t -test.

Article Snippet: The polyclonal rabbit anti-vIRF1 antibody was from Dr. Gary Hayward from Viral Oncology Program, The Johns Hopkins School of Medicine (Baltimore, Maryland, USA) [ – ].

Techniques: Dot Blot, Staining, Control, Northern Blot, Quantitative RT-PCR, Mutagenesis, Over Expression, Plasmid Preparation, Expressing, Western Blot, SDS-Gel, In Vitro, Comparison

Journal: Cell Death and Differentiation

Article Title: Lactylation of NAT10 promotes N 4 ‐acetylcytidine modification on tRNA Ser-CGA-1-1 to boost oncogenic DNA virus KSHV reactivation

doi: 10.1038/s41418-024-01327-0

Figure Lengend Snippet: A Schematic representation of DUF1726 domain (marked in blue), RNA helicase domain (marked in yellow), N -acetyltransferase domain (marked in green) and RNA-binding domain (marked in purple) of NAT10. The identified lactylation lysine site ( K ) within the RNA helicase domain was marked in red, which was mutated to arginine ( R ) and marked in dark blue. B The iSLK-KSHV cells transduced with the wild type (NAT10-Flag), the mutant NAT10 (K290R-Flag) or its control (pCDH) for 48 h were subjected to the anti-Flag immunoprecipitation, and then examined by Western blot with the anti-Pan Kla antibody. C The sequences around NAT10 Lys290 from different species were aligned. Conserved Lys290 was marked in red. D The total RNAs from cells shown as in ( B ) were subjected to dot blot assay with anti-ac 4 C antibody. Methylene blue staining was used as the internal control. E The iSLK-KSHV cells with THUMPD1 overexpression (THUMPD1-Myc) were transduced with the wild type (NAT10-Flag), the mutant NAT10 (K290R-Flag) or its control (pCDH) for 48 h. Cells were subjected to the anti-Flag immunoprecipitation and analyzed by Western blot using anti-Myc antibody. F The total RNAs from iSLK-KSHV cells transduced with the wild type (NAT10) and the mutant (K290R) NAT10 for 48 h were subjected to Northern blot with probes to tRNA Ser-CGA-1-1 , and U6 as the control. G The expression levels of vIRF1 and K-bZIP in cells treated as in ( B ) were examined by Western blot with the corresponding antibodies. H The RT-qPCR analysis for the ribosome-nascent chain-complex-bound mRNA (RNC-qPCR) of representative viral genes of KSHV (RTA, K5, K8, ORF45, ORF57, vIRF1, vIL-6, vBCL-2, ORF65, and K8.1) in cells shown as in ( B ). * P < 0.05, ** P < 0.01 and *** P < 0.001 by Student’s t -test. I The RT-qPCR analysis for the ribosome-nascent chain-complex-bound mRNA (RNC-qPCR) of representative viral genes of KSHV (RTA, K5, K8, vIRF1, vIL-6, vBCL-2, and ORF65) in iSLK-KSHV cells with the wild type (NAT10-WT) or the mutant (NAT10-K290R) NAT10 overexpression, which were complemented with the wild type (tRNA Ser-CGA-1-1 -WT) or the mutant (tRNA Ser-CGA-1-1 -Mut) tRNA Ser-CGA-1-1 for 48 h. * P < 0.05, **, P < 0.01 and *** P < 0.001 by Student’s t -test. J By the assessment of ORF26, real-time DNA-PCR for cells treated as in ( B ) was performed to examine viral copy number after doxycycline stimulation for 72 h. *** P < 0.001 by Student’s t -test.

Article Snippet: The polyclonal rabbit anti-vIRF1 antibody was from Dr. Gary Hayward from Viral Oncology Program, The Johns Hopkins School of Medicine (Baltimore, Maryland, USA) [ – ].

Techniques: RNA Binding Assay, Transduction, Mutagenesis, Control, Immunoprecipitation, Western Blot, Dot Blot, Staining, Over Expression, Northern Blot, Expressing, Quantitative RT-PCR

Journal: Cell Death and Differentiation

Article Title: Lactylation of NAT10 promotes N 4 ‐acetylcytidine modification on tRNA Ser-CGA-1-1 to boost oncogenic DNA virus KSHV reactivation

doi: 10.1038/s41418-024-01327-0

Figure Lengend Snippet: A The expression levels of vIRF1 and K-bZIP in iSLK-KSHV cells transduced with lentiviral sgATAT1 (sgATAT1 #2, sgATAT1 #3) or control lentivirus (Lenti-V2) for 72 h were examined by Western blot with the corresponding antibodies. B The RT-qPCR analysis for the ribosome-nascent chain-complex-bound mRNA (RNC-qPCR) of representative viral genes of KSHV (K5, K8, vIRF1, vIL-6, vBCL-2, and ORF65) in cells treated as in ( A ). * P < 0.05, ** P < 0.01 and *** P < 0.001 by Student’s t -test. C By the assessment of ORF26, real-time DNA-PCR for cells treated as in ( A ) was performed to examine viral copy number after doxycycline stimulation for 72 h. ***, P < 0.001 by Student’s t -test. D The RT-qPCR analysis for the ribosome-nascent chain-complex-bound mRNA (RNC-qPCR) of representative viral genes of KSHV (RTA, K5, K8, ORF45, ORF57, vIRF1, vIL-6, vBCL-2, ORF65, and K8.1) in iSLK-KSHV cells transduced with lentiviral ATAT1 (ATAT1) or its control (pCDH) for 48 h. ***, P < 0.001 by Student’s t -test. E By the assessment of ORF26, real-time DNA-PCR for cells treated as in ( D ) was performed to detect viral copy number after doxycycline stimulation for 72 h. ** P < 0.01 by Student’s t -test.

Article Snippet: The polyclonal rabbit anti-vIRF1 antibody was from Dr. Gary Hayward from Viral Oncology Program, The Johns Hopkins School of Medicine (Baltimore, Maryland, USA) [ – ].

Techniques: Expressing, Transduction, Control, Western Blot, Quantitative RT-PCR

Journal: Science advances

Article Title: XAF1 promotes anti-RNA virus immune responses by regulating chromatin accessibility.

doi: 10.1126/sciadv.adg5211

Figure Lengend Snippet: Fig. 3. XAF1 interacts with MAVS and TBK1. (A) coIP assay analysis of the interaction between endogenous XAF1 and RIG-I, MDA-5, TBK1, MAVS, IRF1, IRF3, and IKKe in HT-29 cells. (B) Diagram detailing the series of truncations of MAVS, TBK1, and XAF1. (C and D) coIP assay analysis shows which domains of MAVS and XAF1 interacted. Red arrowheads in (C) and (D) highlight the interaction bands. (E and F) coIP assay analysis shows which domains of TBK1 and XAF1 interacted. Red arrowheads in (E) and (F) highlight the interaction bands. (G) Direct interaction of XAF1 with MAVS, STING, and TBK1 was detected by a yeast two-hybrid assay. Yeast strain AH109 was transfected with PGBKT7-XAF1, PACT2-MAVS, or PACT2-STING and inoculated on the indicated media for 3 days. ERAL1 and MAVS were introduced as positive controls. Data are representative of three independent experiments with similar results. WCL, whole-cell lysate; IP, immunoprecipitation; IB, immunoblot.

Article Snippet: Rabbit antibodies against MAVS (catalog no. CL488-14341, RRID:AB_2883082), IRF1 (catalog no. CL488-11335, RRID:AB_2919026), Lamin A/C (catalog no. 10298-1-AP, RRID:AB_2296961), KAP1 (catalog no. 15202–1-AP, RRID:AB_2209890), SUMO1 (catalog no. 10329-1-AP, RRID:AB_2286872), SUMO2/3 (catalog no. 11251-1-AP, RRID:AB_2198405), LaminB (catalog no. 66095-1-Ig, RRID:AB_11232208), and mouse antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (catalog no. HRP60004, RRID:AB_2737588) were purchased from Proteintech.

Techniques: Co-Immunoprecipitation Assay, Y2H Assay, Transfection, Immunoprecipitation, Western Blot

Journal: Science advances

Article Title: XAF1 promotes anti-RNA virus immune responses by regulating chromatin accessibility.

doi: 10.1126/sciadv.adg5211

Figure Lengend Snippet: Fig. 5. XAF1 is required for induction of IRF1- and IRF3- target genes. (A) Analysis of IRF1-, IRF3-, and IRF5-regulated gene expression levels in WT and XAF1−/−HT-29 cells from RNA-seq data. (B) coIP assay analysis of the interaction between XAF1 and IRF1, IRF2, IRF3, IRF4, IRF5, IRF6, IRF7, IRF8, and IRF9. (C) Western blot showed protein level of endogenous IRF1 in WT and XAF1−/−HT-29 cells. (D) qRT-PCR analysis of IFNB1 after NT or after stimulation with VSV for 12 hours in WT, XAF1−/−, IRF1−/−, and IRF3−/−HT-29 cells. (E and F) Cytoplasmic and nuclear fractions were isolated, and then, the time course of changes in (E) IRF1 levels and (F) IRF3 levels in each fraction after VSV, SeV, and HSV-1 infection in HT-29 cells was tracked by Western blotting analysis. The relative expression of IRF1 and IRF3 (relative to GAPDH or LMNA) curve shows its dynamic changes in the cytoplasm and nucleus. (G) Relative expression of XAF1, IRF1 and IRF3 (relative to GAPDH or LMNA) curve shows its dynamic changes in the cytoplasm and nucleus after VSV infection in HT-29 cells. (H) qRT-PCR analysis of BST2, RNASEL, OAS2, and DDX60 after NT or after stimulation with VSV for 12 hours in WT and XAF1−/−HT-29 cells. Data are means ± SEM (n = 3) from three independent experiments. *P < 0.05 and **P < 0.01, and ***P < 0.001 [Student’s t test (A), (D), and (H)]. Data are representative of two (A) or three [(B) to (H)] independent experiments with similar results. RPKM, reads per kilobase per million mapped reads; HA, hemagglutinin.

Article Snippet: Rabbit antibodies against MAVS (catalog no. CL488-14341, RRID:AB_2883082), IRF1 (catalog no. CL488-11335, RRID:AB_2919026), Lamin A/C (catalog no. 10298-1-AP, RRID:AB_2296961), KAP1 (catalog no. 15202–1-AP, RRID:AB_2209890), SUMO1 (catalog no. 10329-1-AP, RRID:AB_2286872), SUMO2/3 (catalog no. 11251-1-AP, RRID:AB_2198405), LaminB (catalog no. 66095-1-Ig, RRID:AB_11232208), and mouse antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (catalog no. HRP60004, RRID:AB_2737588) were purchased from Proteintech.

Techniques: Gene Expression, RNA Sequencing, Co-Immunoprecipitation Assay, Western Blot, Quantitative RT-PCR, Isolation, Infection, Expressing

Journal: Science advances

Article Title: XAF1 promotes anti-RNA virus immune responses by regulating chromatin accessibility.

doi: 10.1126/sciadv.adg5211

Figure Lengend Snippet: Fig. 6. XAF1 facilitates the immune responses by inhibiting TRIM28. (A) A flow chart depicting the process coIP experiments and mass spectrometry to detect proteins interacting with XAF1 in the nucleus. (B) GO analysis of the up-regulated proteins immunoprecipitated by XAF1 after VSV infection. Proteins about chromatin remodeling processes were listed. TRIM28 was identified as an XAF1-interacting protein. (C) coIP assay analysis of the interaction between XAF1 and TRIM28. (D and E) coIP assay analysis shows the protein interaction domains between XAF1 and TRIM28. (F) HEK293T cells were transfected with the indicated Vector FL-XAF1 and ZF7- deletion-XAF1 plasmids for 24 hours. After 12 hours of VSV infection, qRT-PCR analyses of IFNB1 mRNA (top) and VSVG mRNA (bottom) were performed. (G) qRT-PCR analysis of IFNB1 after NT or after stimulation with SeV, VSV, or HSV-1 for 12 hours in WT and WT + TRIM28 sgRNA HT-29 cells. (H) qRT-PCR analysis of IRF1-, IRF3-, and NF- κB–regulated genes after NT or after stimulation with VSV for 12 hours in HEK293T cells transiently transfected with Vector, TRIM28 FL, TRIM28 1 to 625, TRIM28 FL + XAF1. Data are means ± SEM (n = 3) from three independent experiments. *P < 0.05 [Student’s t test (F) to (H)]. Data are representative of three [(C) to (H)] independent experiments with similar results.

Article Snippet: Rabbit antibodies against MAVS (catalog no. CL488-14341, RRID:AB_2883082), IRF1 (catalog no. CL488-11335, RRID:AB_2919026), Lamin A/C (catalog no. 10298-1-AP, RRID:AB_2296961), KAP1 (catalog no. 15202–1-AP, RRID:AB_2209890), SUMO1 (catalog no. 10329-1-AP, RRID:AB_2286872), SUMO2/3 (catalog no. 11251-1-AP, RRID:AB_2198405), LaminB (catalog no. 66095-1-Ig, RRID:AB_11232208), and mouse antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (catalog no. HRP60004, RRID:AB_2737588) were purchased from Proteintech.

Techniques: Mass Spectrometry, Immunoprecipitation, Infection, Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Quantitative RT-PCR

Journal: Science advances

Article Title: XAF1 promotes anti-RNA virus immune responses by regulating chromatin accessibility.

doi: 10.1126/sciadv.adg5211

Figure Lengend Snippet: Fig. 7. XAF1 is recruited by IRF1 to inhibit TRIM28. (A) Effects of XAF1 deficiency on endogenous SUMOylation of TRIM28 with or without VSV for 6 hours. (B) Effects of XAF1 deficiency on endogenous SUMOylation of TRIM28 with or without HSV-1 for 6 hours. (C) Western blotting showing the SUMOylation of TRIM28 in HEK293T cells transfected with Vector or XAF1 for 24 hours. (D) Western blotting showing endogenous SUMOylation of TRIM28 in HEK293T cells transfected with Vector, WT-XAF1, S252A-XAF1, or ZF7-deletion-XAF1 for 24 hours. (E) All up-regulated transposons and ERVs after VSV infection in WT HT-29 cells form RNA-seq were showed. Then, these transposons and ERVs change after VSV infection in XAF1−/−HT-29 were also showed. (F) coIP assay analysis of the interaction between XAF1 and TRIM28 in WT HT-29 cells compared with IRF1−/−HT-29 cells with or without VSV infection. (G and H) WT and IRF1−/−HT-29 cells transfected with HA-XAF1 were NT or infected VSV for 3 hours, and ChIP-qPCR signal showing HA-XAF1 (G) or H3K27Ac (H) occupancy of indicated genes. Data are means ± SEM (n = 3) from three independent experiments. *P < 0.05 [Student’s t test (F) to (H)]. Data are representative of three [(A) to (G)] independent experiments with similar results. H3K27ac, H3 lysine 27 acetylation.

Article Snippet: Rabbit antibodies against MAVS (catalog no. CL488-14341, RRID:AB_2883082), IRF1 (catalog no. CL488-11335, RRID:AB_2919026), Lamin A/C (catalog no. 10298-1-AP, RRID:AB_2296961), KAP1 (catalog no. 15202–1-AP, RRID:AB_2209890), SUMO1 (catalog no. 10329-1-AP, RRID:AB_2286872), SUMO2/3 (catalog no. 11251-1-AP, RRID:AB_2198405), LaminB (catalog no. 66095-1-Ig, RRID:AB_11232208), and mouse antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (catalog no. HRP60004, RRID:AB_2737588) were purchased from Proteintech.

Techniques: Western Blot, Transfection, Plasmid Preparation, Infection, RNA Sequencing, Co-Immunoprecipitation Assay, ChIP-qPCR

Journal: Science advances

Article Title: XAF1 promotes anti-RNA virus immune responses by regulating chromatin accessibility.

doi: 10.1126/sciadv.adg5211

Figure Lengend Snippet: Fig. 8. Model: XAF1 promotes innate immune response against RNA viruses by regulating chromatin accessibility. Upon RNA virus infection, XAF1 is recruited by MAVS and then phosphorylated by TBK1 at serine-252, which initiates its translocation from the cytoplasm to the nucleus. XAF1 then interacted with IRF1 and specifically liberated the IRF1-targeted chromatin by blocking the SUMOylation of the chromatin repressor TRIM28. This results in the desuppression of chromatin to enhance the transcription of IRF1-regulated innate immune genes to initiate IFN-independent innate immune responses. On the other hand, XAF1 and IRF1 promotes opening of chromatin targeted by IRF3 to initiate an IFN-dependent innate immune response.

Article Snippet: Rabbit antibodies against MAVS (catalog no. CL488-14341, RRID:AB_2883082), IRF1 (catalog no. CL488-11335, RRID:AB_2919026), Lamin A/C (catalog no. 10298-1-AP, RRID:AB_2296961), KAP1 (catalog no. 15202–1-AP, RRID:AB_2209890), SUMO1 (catalog no. 10329-1-AP, RRID:AB_2286872), SUMO2/3 (catalog no. 11251-1-AP, RRID:AB_2198405), LaminB (catalog no. 66095-1-Ig, RRID:AB_11232208), and mouse antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (catalog no. HRP60004, RRID:AB_2737588) were purchased from Proteintech.

Techniques: Virus, Infection, Translocation Assay, Blocking Assay