Journal: Cell reports

Article Title: PARP7 inhibition stabilizes STAT1/STAT2 and relieves experimental autoimmune encephalomyelitis in mice

doi: 10.1016/j.celrep.2025.116130

Figure Lengend Snippet: (A) Immunoblot analysis of STAT1 and STAT2 protein levels in PARP7 WT and KO MEF cells. The experiment was repeated twice. (B) Co-immunoprecipitation (coIP) analysis showing that STAT1 and STAT2 interact with PARP7. FLAG-PARP7 was ectopically expressed in HEK293T cells. FLAG-PARP7 was immunoprecipitated and then blotted for endogenous STAT1 and STAT2. The experiment was repeated twice. (C and D) Immunoblot analysis of ADP-ribosylated STAT1 (C) and STAT2 (D) in PARP7 WT and KO MEF cells. Cells were lysed in the presence of biotin-NAD + or NAD + . Proteins that were ADP-ribosylated by biotin-NAD + were pulled down with streptavidin beads and then blotted for STAT1 or STAT2. Samples treated with NAD + were used as a negative control. Both experiments were repeated three times. (E) PARP7 WT, but not the H532A mutant, ADP-ribosylates STAT1. GFP-tagged empty vector (EV), WT PARP7, or the H532 mutant were co-transfected with FLAG-STAT1 into HEK293T cells. FLAG-STAT1 was pulled down to detect interaction with PARP7 (blotted with GFP antibody) and ADP-ribosylation (blotted with ADP-ribose antibody). The experiment was repeated twice. See also .

Article Snippet: STAT1 (9172), phospho-STAT1 (Tyr701) (D4A7) (7649), STAT2 (D9J7L) (72604), phospho-Stat2 (Tyr690) (# 4441), ubiquitin (P4D1) (# 3936), TBK1/NAK (3013), phospho-TBK1/NAK (Ser172) (D52C2) (# 5483), SQSTM1/p62 (# 5114), Poly/Mono-ADP Ribose (D9P7Z) (# 89190), JAK1 (6G4) (# 3344), α-Tubulin (# 2144) antibodies were purchased from Cell Signaling.

Techniques: Western Blot, Immunoprecipitation, TNKS1 Histone Ribosylation Assay, Negative Control, Mutagenesis, Plasmid Preparation, Transfection

Journal: Cell reports

Article Title: PARP7 inhibition stabilizes STAT1/STAT2 and relieves experimental autoimmune encephalomyelitis in mice

doi: 10.1016/j.celrep.2025.116130

Figure Lengend Snippet: (A) HEK293T cells were transfected with FLAG-WT or H532A mutant PARP7 overnight. FLAG-PARP7 was detected by immunofluorescence with anti-FLAG (green) antibodies. Endogenous STAT1 was detected by immunofluorescence with Alexa Flour 647. Nuclei were stained with Hoechst stain (blue) (scale bar: 2 μm). Right: quantification of STAT1 and PARP7 co-localization using Pearson’s correlation coefficient. Data are presented as mean ± SD. *** p < 0.001, unpaired t test. (B) HEK293T cells were transfected with FLAG-WT or H532A mutant PARP7 overnight. FLAG-PARP7 was detected by immunofluorescence with anti-FLAG (green) antibodies. Endogenous STAT2 was detected by immunofluorescence with Alexa Flour 647. Nuclei were stained with Hoechst stain (blue) (scale bar: 2 μm.). Right: quantification of STAT2 and PARP7 co-localization using Pearson’s correlation coefficient. Data are presented as mean ± SD. *** p < 0.001, unpaired t test. All experiments were repeated twice. See also .

Article Snippet: STAT1 (9172), phospho-STAT1 (Tyr701) (D4A7) (7649), STAT2 (D9J7L) (72604), phospho-Stat2 (Tyr690) (# 4441), ubiquitin (P4D1) (# 3936), TBK1/NAK (3013), phospho-TBK1/NAK (Ser172) (D52C2) (# 5483), SQSTM1/p62 (# 5114), Poly/Mono-ADP Ribose (D9P7Z) (# 89190), JAK1 (6G4) (# 3344), α-Tubulin (# 2144) antibodies were purchased from Cell Signaling.

Techniques: Transfection, Mutagenesis, Immunofluorescence, Staining

Journal: Cell reports

Article Title: PARP7 inhibition stabilizes STAT1/STAT2 and relieves experimental autoimmune encephalomyelitis in mice

doi: 10.1016/j.celrep.2025.116130

Figure Lengend Snippet: (A) Western blot analysis of STAT1 and STAT2 in PARP7 WT or KO MEF cells after treatment of MG132 at 20 μМ (left) and cycloheximide (CHX) at 50 μМ (right). The experiment was repeated three times. (B) Western blot analysis of STAT1 and STAT2 in PARP7 WT or KO MEF cells after bafilomycin A1 treatment at 5 μМ. The experiment was repeated three times. (C) coIP analysis of p62 and PARP7 WT or H532A catalytic mutant. FLAG-tagged PARP7 WT and H532A mutant were transfected into HEK293T cells, and coIP was performed to detect interactions between p62 and PARP7 WT or H532A mutant. The experiment was repeated three times. (D) Co-localization of hemagglutinin (HA)-tagged p62 with GFP-tagged PARP7 WT or H532A mutant in HEK293T cells. HA-p62 was detected by immunofluorescence with anti-HA (red) antibody. Nuclei were stained with Hoechst stain (scale bar: 5 μm). The experiment was repeated twice. (E) PARP7 promotes STAT1 and STAT2 ubiquitination. HEK293T cells were co-transfected with FLAG-STAT1 (left) or FLAG-STAT2 (right) and GFP-tagged PARP7 WT or H532A mutant. STAT1 or STAT2 was immunoprecipitated by anti-FLAG affinity resins, and the ubiquitination of STAT1 or STAT2 was analyzed by western blot. The experiment repeated twice. (F) PARP7 promotes p62 interaction with STAT1 and STAT2. HEK293T cells were co-transfected with FLAG-STAT1 (left) or FLAG-STAT2 (right) and GFP-tagged PARP7 WT or H532A mutant. STAT1 or STAT2 was immunoprecipitated by anti-FLAG affinity resin and blotted for p62. The experiment was repeated twice. (G) p62 knockdown diminishes the difference in STAT1 and STAT2 levels between PARP7 WT and KO MEF cells. STAT1 and STAT2 protein levels in PARP7 WT and KO MEF cells with or without p62 knockdown were analyzed by western blots. The experiment was repeated twice. (H) Proposed model depicting the negative regulation of STAT1 and STAT2 by PARP7. PARP7 binds and ADP-ribosylates STAT1 and STAT2. The ADP-ribosylation recruits E3 ubiquitin ligases, leading to the ubiquitination of STAT1 and STAT2. The ubiquitinated STAT1 and STAT2 then recruits p62, leading to autophagy-mediated degradation. Created with BioRender.com . See also – .

Article Snippet: STAT1 (9172), phospho-STAT1 (Tyr701) (D4A7) (7649), STAT2 (D9J7L) (72604), phospho-Stat2 (Tyr690) (# 4441), ubiquitin (P4D1) (# 3936), TBK1/NAK (3013), phospho-TBK1/NAK (Ser172) (D52C2) (# 5483), SQSTM1/p62 (# 5114), Poly/Mono-ADP Ribose (D9P7Z) (# 89190), JAK1 (6G4) (# 3344), α-Tubulin (# 2144) antibodies were purchased from Cell Signaling.

Techniques: Western Blot, Mutagenesis, Transfection, Immunofluorescence, Staining, Ubiquitin Proteomics, Immunoprecipitation, Knockdown

Journal: Scientific Reports

Article Title: STAT1 is essential for the inhibition of hepatitis C virus replication by interferon-λ but not by interferon-α

doi: 10.1038/srep38336

Figure Lengend Snippet: ( A ) STAT2 knockout (KO) clones #1 and #2 were established from Huh-7.5 cells expressing a STAT2 sgRNA. DNA and amino acid sequences surrounding the sgRNA target sequences (blue) are shown. The protospacer adjacent motif (PAM) and the mutation in each allele are shown in green and red, respectively. ( B ) NT sgRNA-expressing cells and STAT2 KO cells (clones #1 and #2) were infected with HCVcc and treated with IFN-α (1,000 U/ml) for the indicated times. The expression levels of NS5A, PKR, and STAT2 were evaluated by immunoblotting (IB). ( C ) NT sgRNA-expressing cells and STAT2 KO cells were infected with HCVcc and treated with IFN-α (1,000 U/ml) for 96 h. Intracellular HCV RNA levels were quantified by real-time PCR and normalized to control values. Data represent the mean ± S.D. ( n = 3). * P < 0.01. NS, not significant.

Article Snippet: Anti-phospho-STAT2 (600-401-A93S) antibody was purchased from Rockland Immunochemicals.

Techniques: Knock-Out, Clone Assay, Expressing, Mutagenesis, Infection, Western Blot, Real-time Polymerase Chain Reaction, Control

Journal: Scientific Reports

Article Title: STAT1 is essential for the inhibition of hepatitis C virus replication by interferon-λ but not by interferon-α

doi: 10.1038/srep38336

Figure Lengend Snippet: ( A , B ) STAT3 knockout (KO) clones #1, #2 were established from Huh-7.5 cells expressing STAT3 sgRNAs #1 and #2, respectively. STAT6 knockout (KO) clones #1 and #2 were established from Huh-7.5 cells expressing STAT6 sgRNAs #1 and #2, respectively. ( A ) Cells were infected with HCVcc and treated with IFN-α (1,000 U/ml) for 72 h. The expression levels of NS5A, PKR, IRF9, STAT1, STAT2, STAT3, and STAT6 were evaluated by immunoblotting (IB). ( B ) Cells were infected with HCVcc and treated with IFN-α (1,000 U/ml) for 96 h. Intracellular HCV RNA levels were quantified by real-time PCR and normalized to control values. Data represent the mean ± S.D. ( n = 3). * P < 0.01. NS, not significant.

Article Snippet: Anti-phospho-STAT2 (600-401-A93S) antibody was purchased from Rockland Immunochemicals.

Techniques: Knock-Out, Clone Assay, Expressing, Infection, Western Blot, Real-time Polymerase Chain Reaction, Control

Journal: Scientific Reports

Article Title: STAT1 is essential for the inhibition of hepatitis C virus replication by interferon-λ but not by interferon-α

doi: 10.1038/srep38336

Figure Lengend Snippet: ( A ) NT sgRNA-expressing Huh-7.5 cells, STAT1 knockout (KO) cells (clones #1 and #2), and STAT2 KO cells (clone #1) were infected with HCVcc and treated with IFN-λ (1,000 U/ml) for the indicated times. The expression levels of NS5A, PKR, STAT1, and STAT2 were evaluated by immunoblotting (IB). ( B ) NT sgRNA-expressing cells, STAT1 KO cells (clones #1 and #2), and STAT2 KO cells (clone #1) were infected with HCVcc and treated with IFN-λ (1,000 U/ml) for 96 h. Intracellular HCV RNA levels were quantified by real-time PCR and normalized to control values. Data represent the mean ± S.D. ( n = 3). * P < 0.01. NS, not significant.

Article Snippet: Anti-phospho-STAT2 (600-401-A93S) antibody was purchased from Rockland Immunochemicals.

Techniques: Expressing, Knock-Out, Clone Assay, Infection, Western Blot, Real-time Polymerase Chain Reaction, Control

Journal: Scientific Reports

Article Title: STAT1 is essential for the inhibition of hepatitis C virus replication by interferon-λ but not by interferon-α

doi: 10.1038/srep38336

Figure Lengend Snippet: ( A ) NT sgRNA-expressing Huh-7.5 cells and STAT1 knockout (KO) cells (clone #2) were infected with HCVcc and treated with IFN-α (1,000 U/ml) or IFN-λ (1,000 U/ml) for the indicated times. The expression levels of NS5A, PKR, IRF9, STAT1, and STAT2 were evaluated by immunoblotting (IB). ( B ) NT sgRNA-expressing cells, STAT1 KO cells (clones #1 and #2), and STAT2 KO cells (clone #1) were treated with IFN-α (1,000 U/ml) or IFN-λ (1,000 U/ml) for the indicated times. The levels of PKR and MX1 mRNAs were evaluated by real-time PCR and normalized to control values. Data represent the mean ± S.D. ( n = 3). ( C ) NT sgRNA-expressing cells and STAT1 KO cells (clone #1) were treated with IFN-α (1,000 U/ml) or IFN-λ (1,000 U/ml) for 24 h. Microarray analysis was performed. Fold changes relative to untreated NT sgRNA-expressing cells were calculated. Probe sets that showed >1.5-fold increase in response to both IFN-α and IFN-λ in NT sgRNA-expressing cells but showed little changes (within 1.5-fold) due to STAT1 KO were selected. Heat maps were generated using the microarray data. See for a full list of the selected probe sets and fold changes.

Article Snippet: Anti-phospho-STAT2 (600-401-A93S) antibody was purchased from Rockland Immunochemicals.

Techniques: Expressing, Knock-Out, Infection, Western Blot, Clone Assay, Real-time Polymerase Chain Reaction, Control, Microarray, Generated

Journal: Scientific Reports

Article Title: STAT1 is essential for the inhibition of hepatitis C virus replication by interferon-λ but not by interferon-α

doi: 10.1038/srep38336

Figure Lengend Snippet: ( A , B ) NT sgRNA-expressing Huh-7.5 cells, STAT1 knockout (KO) cells (clones #1 and #2), and STAT2 KO cells (clone #1) were treated with IFN-α (1,000 U/ml) ( A ) or IFN-λ (1,000 U/ml) ( B ) for the indicated times. Phosphorylation of STAT1 and STAT2 was evaluated by immunoblotting (IB).

Article Snippet: Anti-phospho-STAT2 (600-401-A93S) antibody was purchased from Rockland Immunochemicals.

Techniques: Expressing, Knock-Out, Clone Assay, Phospho-proteomics, Western Blot

Journal: Immunology

Article Title: Interferon-α promotes MHC I antigen presentation of islet β cells through STAT1-IRF7 pathway in type 1 diabetes.

doi: 10.1111/imm.13468

Figure Lengend Snippet: FIGURE 6 Inhibition of STAT1 and STAT2 prevented IFN-α induced MHC-I and antigen presentation in MIN6 cells. MIN6 cells were transfected with siRNA (si-STAT1 or/and si-STAT2) and then treated with or without IFN-α (1000 U/ml) for 48 h. (a) Protein and (b) mRNA of antigen presented molecules was measured. (c) Representative images (10×) of MHC I in MIN6 cells after IFN-α and siRNA treatment. *p < 0·05, **p < 0·01 and ***p < 0·001

Article Snippet: Phospho- STAT1, STAT1, Phospho- STAT2, STAT2, TAP1 and β- actin antibodies were purchased from Cell Signaling Technology company, USA; IRF7 antibody was purchased from NOVUS company, USA; B2M, PSMB8 and NLRC5 antibodies were purchased from Abcam company, UK; PhosphoSTAT3, STAT3 and Phospho- STAT5 antibodies were purchased from Abclonal company, China; STAT5 and IRF9 antibodies were purchased from company, China.

Techniques: Inhibition, Immunopeptidomics, Transfection

Journal: Immunology

Article Title: Interferon-α promotes MHC I antigen presentation of islet β cells through STAT1-IRF7 pathway in type 1 diabetes.

doi: 10.1111/imm.13468

Figure Lengend Snippet: FIGURE 8 IFN-α induce β cells autoimmunity by promoting MHC I antigen presentation. In β cells, IFN-α binds to the IFNRs and lead to the nuclear translocation of STAT1 and IRF7, which in turn activates the STAT1-IRF7 axis to increase expression of antigen presenting molecules (TAP1, PSMB8 and MHC I). These actions create positive feedback through IRF7-STAT2 cascade amplifying signals and promote the proliferation of CD8+ T cells and insulitis. Figure was created with BioRender.com (Agreement number: TX23NA2XMH)

Article Snippet: Phospho- STAT1, STAT1, Phospho- STAT2, STAT2, TAP1 and β- actin antibodies were purchased from Cell Signaling Technology company, USA; IRF7 antibody was purchased from NOVUS company, USA; B2M, PSMB8 and NLRC5 antibodies were purchased from Abcam company, UK; PhosphoSTAT3, STAT3 and Phospho- STAT5 antibodies were purchased from Abclonal company, China; STAT5 and IRF9 antibodies were purchased from company, China.

Techniques: Immunopeptidomics, Translocation Assay, Expressing

Journal: bioRxiv

Article Title: ISG15 orchestrates dynamic crosstalk between mitochondrial fat oxidation and type 1 interferon in myeloid cells

doi: 10.64898/2026.01.19.700051

Figure Lengend Snippet: (A ) Strategy for CRISPR Cas9 mediated genetic depletion of Cpt1a in primary BMDMs. (B) Seahorse analysis of OCR in Cpt1a KO and control BMDMs on DMXAA stimulation on sequential treatment with oligomycin, FCCP and Rotenone/Antimycin A (n=8 per group). (C-D) Histograms of basal OCR (C) and maximal respiration (D) of seahorse analysis in B . (E) Histograms showing IFN-β cytokine levels in Cpt1a KO and control BMDMs (n=4 per group) on treatment with DMXAA for indicated time points (0-6h). Results measured were normalized to the cell counts in each corresponding wells using two-way ANOVA followed by Tukey’s multiple comparisons test. (F) Representative immunoblots of pSTAT1, STAT1, p-STAT2, STAT2, ISG15, p-IRF3, IRF3 and vinculin in DMXAA treated control and Cpt1a KO BMDMs upon treatment with DMXAA for indicated time points (0-6h) (n=3 per group). (G) Representative blots of Ac-His3 K9/K14, total His3 and vinculin in control and Cpt1a KO BMDMs upon treatment with DMXAA for indicated time points (0-6h) (n=3 per group). (H) Schematic of proposed epigenetic control whereby type 1 IFN in a feed forward manner increases further interferon production in response by increaing myeloid cell mitochondrial FAO to generate Acetyl-CoA and histone acetylation. Asterisks represent - * p < 0.05; ** p < 0.01; *** p < 0.001. n.s. - not significant.

Article Snippet: The following antibodies were used: STAT1, phospho-STAT1, STAT2, phospho-IRF3, ISG15, acetyl-Histone H3(Lys9/Lys14), Histone H3 (Cell Signaling Technology); phospho-STAT2 (EMD Millipore); IRF3 (Abcam); Cpt1a, HADHB (proteintech); HADHA (Invitrogen); ACAT1 and vinculin (Sigma-Aldrich).

Techniques: CRISPR, Control, Western Blot

Journal: bioRxiv

Article Title: ISG15 orchestrates dynamic crosstalk between mitochondrial fat oxidation and type 1 interferon in myeloid cells

doi: 10.64898/2026.01.19.700051

Figure Lengend Snippet: (A ) Schematic of the murine infection study design. LCMV infected C57bl/6 mice were sacrificed at indicated time points (0,1,3,5 and 8 days) post infection and splenocytes were isolated (n=5 mice per group/day). CD11b+ myeloid cells were separated from the splenocytes and used for this study. (B-G) Histograms showing quantitative RT-PCR analysis of type 1 IFN response genes Ifnb (B) , Stat1 (C) , Stat2 (D) , Isg15 (E) and mitochondrial FAO enzymes Acat1 (F) and Cpt1a (G) in CD11b+ myeloid cells isolated from the splenocytes of infected mice. Data were normalized to 18 S rRNA and represented as means ± SEM. Splenocytes from infected mice were subjected to treatment with 5µM FaO Blue followed by staining with PE conjugated CD11b+ antibody. (H) Representative data of flow cytometry based measurement of fluorescence intensity of FaO Blue in CD11b+ splenocytes from infected mice at indicated time points (J) Representation of flow cytometric geometric mean fluorescence intensity of FaO Blue in infected mice at different timepoints. (K) Representative microscopic images of CD11b+ myeloid cells, isolated from the splenocytes of infected mice at different timepoints, incubated with FaO Blue. (I) Histograms of fluorescence intensity of FaO Blue from microscopic images in I . Data represented as means ± SEM. Two-way ANOVA followed by Tukey’s multiple comparisons test. Asterisks represent * p < 0.05; ** p < 0.01; *** p < 0.001. n.s. - not significant.

Article Snippet: The following antibodies were used: STAT1, phospho-STAT1, STAT2, phospho-IRF3, ISG15, acetyl-Histone H3(Lys9/Lys14), Histone H3 (Cell Signaling Technology); phospho-STAT2 (EMD Millipore); IRF3 (Abcam); Cpt1a, HADHB (proteintech); HADHA (Invitrogen); ACAT1 and vinculin (Sigma-Aldrich).

Techniques: Infection, Isolation, Quantitative RT-PCR, Staining, Flow Cytometry, Fluorescence, Incubation

Journal: bioRxiv

Article Title: ISG15 orchestrates dynamic crosstalk between mitochondrial fat oxidation and type 1 interferon in myeloid cells

doi: 10.64898/2026.01.19.700051

Figure Lengend Snippet: (A ) Representative immunoblots of p-STAT1, STAT1, p-STAT2, STAT2, p-IRF3, IRF3 and vinculin from control and Isg15 KO BMDMs stimulated with DMXAA for different timepoints (0-12h).(n=5) (B) Volcano plot representation of differentially expressed genes in IFN-α treated PBMCs from ISG15 deficient patients compared to IFN-α treated PBMCs from healthy volunteers (GSE60359). x-axis represents log2 fold change in gene expression, and y-axis represents the −log10 adjusted p-value. Each point corresponds to a gene. Genes with large fold changes and high statistical significance appear toward the top left and top right of the plot, highlighting significantly downregulated and upregulated genes, respectively. Genes associated with type 1 IFN response has been highlighted as yellow dots. (C-E) Dot plot representation of scRNA analysis to understand the expression pattern of FAO associated genes in different immune cells from dermal tissues of healthy volunteers (C) , DLE (D) and SLE (E) patients. Dot size represents the percentage of immune cells expressing each gene and dot color represents the average expression level of indicated genes. (F) Schematic of the clinical validation study to determine FAO in CD14+ myeloid cells from PBMCs of control vs. SLE patients. PBMCs isolated from blood of 9 SLE patients and age matched healthy volunteers were incubated with 5 µM FaO Blue for 1h followed by staining with APC conjugated anti CD14 antibody. The CD14+ populations were gated and analyzed for fluorescent intensity of FaO blue by flow cytometry. (G) Representative data of flow cytometry based measurement of FaO Blue intensity in CD14+ gated PBMCs from SLE patient and respective age matched healthy volunteer. (H) Dot plot of paired geometric mean fluorescent intensity of FaO Blue in CD14+ gated PBMCs from SLE patient and respective age matched healthy volunteer (n=9). (I-M) Quantitative RT-PCR analysis of ISG15 (I) , CPT1A (J) , ACAT1 (K) , HADHA (L) and HADHB (M) in CD14+ myeloid cells isolated from PBMCs of SLE patients and respective age matched healthy volunteers. Data were normalized to 18 S rRNA. Paired t-test. (N) The heatmap shows expression profiling by array data from the GSE60359 dataset, comparing healthy controls and ISG15-deficient patients. Rows represent the genes of key FAO enzymes CRAT , ECHS1 , HADHA , ACAT1 , CPT1A , HADHB , PPARG , and PPARA , while columns represent six individual samples. Expression values were normalized using quantile normalization and scaled to z-scores. Colors indicate relative gene expression levels, as shown in the color bar. Asterisks represent - * p < 0.05; ** p < 0.01; *** p < 0.001. n.s. - not significant.

Article Snippet: The following antibodies were used: STAT1, phospho-STAT1, STAT2, phospho-IRF3, ISG15, acetyl-Histone H3(Lys9/Lys14), Histone H3 (Cell Signaling Technology); phospho-STAT2 (EMD Millipore); IRF3 (Abcam); Cpt1a, HADHB (proteintech); HADHA (Invitrogen); ACAT1 and vinculin (Sigma-Aldrich).

Techniques: Western Blot, Control, Gene Expression, Expressing, Biomarker Discovery, Isolation, Incubation, Staining, Flow Cytometry, Quantitative RT-PCR