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: FEBS Open Bio

Article Title: TRIM66 promotes malignant progression of prostate carcinoma through the JAK/STAT pathway

doi: 10.1002/2211-5463.12798

Figure Lengend Snippet: TRIM66 regulated STAT2 expression. (A) The correlation of TRIM66 and STAT2 in patients with prostate cancer was analyzed in TCGA prostate cancer dataset. (B) The mRNA expression of STAT2 in PC‐3 cells transfected with TRIM66 shRNA was determined by RT‐qPCR. (C) The protein expression of STAT2 and phosphorylated (p)‐STAT2 in PC‐3 cells transfected with TRIM66 shRNA was analyzed using western blot. (D) Luciferase reporter assay of STAT2 promoter in cells transfected with vector or shTRIM66. (E) The mRNA levels of STAT2 in PC‐3 cells transfected with shSTAT2 or corresponding vector were confirmed by RT‐qPCR. (F) Cell viabilities of PC‐3 cells transfected with shSTAT2 or corresponding vector were determined by cell count assay. (G) Fluorescence‐activated cell sorting analysis of the effects of STAT2 depletion on the cell cycle in PC‐3 cells. (H) Transwell of migration and invasion assay of PC‐3 cells transfected with shSTAT2 and corresponding vector. (I) The expression of EMT markers in PC‐3 cells transfected with shSTAT2 and corresponding vector was determined by western blot. All of the experiments were biologically repeated at least three times. Data were expressed as mean ± SD and analyzed by Student’s t ‐test. ** P < 0.01, *** P < 0.001.

Article Snippet: Immunoblotting was performed with the following antibodies: rabbit anti‐STAT2 (ab233177, 1 : 1000), rabbit anti‐STAT2 (phosphor‐Y690; ab205718, 1 : 1000), rabbit anti‐IL‐2 (ab180780, 1 : 1000), rabbit anti‐GAPDH (1 : 1000), and goat anti‐(rabbit horseradish peroxidase) (ab6721, 1 : 5000; Abcam, Cambridge, UK).

Techniques: Expressing, Transfection, shRNA, Quantitative RT-PCR, Western Blot, Luciferase, Reporter Assay, Plasmid Preparation, Cell Counting, Fluorescence, FACS, Migration, Invasion Assay

Journal: FEBS Open Bio

Article Title: TRIM66 promotes malignant progression of prostate carcinoma through the JAK/STAT pathway

doi: 10.1002/2211-5463.12798

Figure Lengend Snippet: TRIM66 regulated IL‐2 expression. (A) The correlation of STAT2 and IL‐2 in patients with prostate cancer was analyzed in TCGA prostate cancer dataset. (B) The protein expression of IL‐2 in PC‐3 cells transfected with STAT2 shRNA was analyzed using western blot. (C) The correlation of TRIM66 and IL‐2 in patients with prostate cancer was analyzed in TCGA prostate cancer dataset. (D) The mRNA expression of IL‐2 in PC‐3 cells transfected with TRIM66 shRNA was determined by RT‐qPCR. (E) The protein expression of IL‐2 in PC‐3 cells transfected with TRIM66 shRNA was analyzed using western blot. (F) The mRNA levels of PC‐3 cells transfected with shIL‐2 or corresponding vector were confirmed by RT‐qPCR. (G) Cell viabilities of PC‐3 cells transfected with shIL‐2 or corresponding vector were determined by cell count assay. (H) Transwell of migration and invasion assay of PC‐3 cells transfected with shIL‐2 or corresponding vector. All of the experiments were biologically repeated at least three times. Data were expressed as mean ± SD and analyzed by Student’s t ‐test. * P < 0.05, ** P < 0.01.

Article Snippet: Immunoblotting was performed with the following antibodies: rabbit anti‐STAT2 (ab233177, 1 : 1000), rabbit anti‐STAT2 (phosphor‐Y690; ab205718, 1 : 1000), rabbit anti‐IL‐2 (ab180780, 1 : 1000), rabbit anti‐GAPDH (1 : 1000), and goat anti‐(rabbit horseradish peroxidase) (ab6721, 1 : 5000; Abcam, Cambridge, UK).

Techniques: Expressing, Transfection, shRNA, Western Blot, Quantitative RT-PCR, Plasmid Preparation, Cell Counting, Migration, Invasion Assay

Journal: FEBS Open Bio

Article Title: TRIM66 promotes malignant progression of prostate carcinoma through the JAK/STAT pathway

doi: 10.1002/2211-5463.12798

Figure Lengend Snippet: TRIM66 regulated prostate cancer cell proliferation and metastasis through mediating the STAT2–IL‐2 axis. (A) The mRNA expression of STAT2 in PC‐3 cells transfected with STAT2 overexpression plasmid was determined by RT‐qPCR. (B) Cell viability of PC‐3 cells transfected with shTRIM66 and/or STAT2 overexpression plasmid was determined by cell counts assay. (C) Transwell of migration and invasion assay of PC‐3 cells transfected with shTRIM66 and/or STAT2 overexpression plasmid. (D) The mRNA expression of IL‐2 in PC‐3 cells transfected with IL‐2 overexpression plasmid was determined by RT‐qPCR. (E) Cell viability of PC‐3 cells transfected with shTRIM66 and/or IL‐2 overexpression plasmid was determined by cell counts assay. (F) Transwell of migration and invasion assay of PC‐3 cells transfected with shTRIM66 and/or IL‐2 overexpression plasmid. (G) Schematic model of TRIM66‐mediated STAT2–IL‐2 signaling axis in prostate cancer cells. All of the experiments were biologically repeated at least three times. Data were expressed as mean ± SD. (A, D) Data were analyzed by Student’s t ‐test, and others by one‐way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Immunoblotting was performed with the following antibodies: rabbit anti‐STAT2 (ab233177, 1 : 1000), rabbit anti‐STAT2 (phosphor‐Y690; ab205718, 1 : 1000), rabbit anti‐IL‐2 (ab180780, 1 : 1000), rabbit anti‐GAPDH (1 : 1000), and goat anti‐(rabbit horseradish peroxidase) (ab6721, 1 : 5000; Abcam, Cambridge, UK).

Techniques: Expressing, Transfection, Over Expression, Plasmid Preparation, Quantitative RT-PCR, Migration, Invasion Assay

Journal: The Journal of Biological Chemistry

Article Title: The NSs protein of severe fever with thrombocytopenia syndrome virus differentially inhibits the type 1 interferon response among animal species

doi: 10.1016/j.jbc.2023.104819

Figure Lengend Snippet: Suppression of STAT1 and STAT2 phosphorylation by NSs. A , HEK293T (human), NIH3T3 (mouse), CRFK (cat), A72 (dog), Mpf (ferret), or PK15 (pig) cells transfected with expression plasmid for HA-tagged NSs were treated with IFN-αA/D (2000 U/ml) or left untreated for 30 min and were then lysed for detection of each protein expression by immunoblotting. B and C , HEK293T (human), NIH3T3 (mouse), CRFK (cat), A72 (dog), Mpf (ferret), or PK15 (pig) cells were infected with SFTSV at an MOI of 10. C , after 18 h or 48 h, these cells were treated with IFN-αA/D (2000 U/ml) or left untreated for 30 min and were then lysed for detection of each protein expression by immunoblotting. The relative expression rates of pSTAT1 and pSTAT2 in mock-transfected cells and uninfected cells treated with IFN-I are set at 100%. The assays were independently performed in triplicate. The data represent averages with SDs. CRFK, Crandell–Rees feline kidney; HA, hemagglutinin; HEK293T, human embryonic kidney 293T cell line; IFN, interferon; MOI, multiplicity of infection; NSs, nonstructural protein; SFTSV, severe fever with thrombocytopenia syndrome virus; STAT, signal transducer and activator of transcription.

Article Snippet: Western blotting was performed as previously described using the following antibodies ( , ): anti-HA (catalog no.: 18850; QED Biosciences, Inc), anti-His (catalog no.: 9F2; Wako), anti-FLAG (catalog no.: F7425; Sigma–Aldrich), anti-STAT2 (catalog no.: D9J7L; Cell Signaling Technology), anti-STAT1 (catalog no.: D19KY; Cell Signaling Technology), anti-STAT2 (phosphor Y690) (catalog no.: GTX50721; GeneTex), anti-STAT1 (phosphor Y701) (catalog no.: D4A7; Cell Signaling Technology), or anti-β-actin (catalog no.: AC-15; Sigma–Aldrich).

Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot, Infection

Journal: The Journal of Biological Chemistry

Article Title: The NSs protein of severe fever with thrombocytopenia syndrome virus differentially inhibits the type 1 interferon response among animal species

doi: 10.1016/j.jbc.2023.104819

Figure Lengend Snippet: Influence of NSs to STAT1 and STAT2 activation in cells derived from different animal species. HEK293T (human), NIH3T3 (mouse), CRFK (cat), A72 (dog), Mpf (ferret), or PK15 (pig) cells were transfected with the expression plasmid for HA-tagged NSs and treated with IFN-αA/D (2000 U/ml) for 30 min at 48 h post-transfection. IFA was performed to detect NSs, the nuclei, and pSTAT1 or pSTAT2, shown in red , blue , and green , respectively. Each figure in the red frame and blue frame indicates the result of indirect IFA with anti-pSTAT1 and anti-pSTAT2, respectively. The a rrow and arrowhead indicate NSs-expressing and nonexpressing cells, respectively. Scale bar represents 50 μm. Representative results of IFA are shown. CRFK, Crandell–Rees feline kidney; HA, hemagglutinin; HEK293T, human embryonic kidney 293T cell line; IFA, immunofluorescence assay; IFN, interferon; NSs, nonstructural protein; STAT, signal transducer and activator of transcription.

Article Snippet: Western blotting was performed as previously described using the following antibodies ( , ): anti-HA (catalog no.: 18850; QED Biosciences, Inc), anti-His (catalog no.: 9F2; Wako), anti-FLAG (catalog no.: F7425; Sigma–Aldrich), anti-STAT2 (catalog no.: D9J7L; Cell Signaling Technology), anti-STAT1 (catalog no.: D19KY; Cell Signaling Technology), anti-STAT2 (phosphor Y690) (catalog no.: GTX50721; GeneTex), anti-STAT1 (phosphor Y701) (catalog no.: D4A7; Cell Signaling Technology), or anti-β-actin (catalog no.: AC-15; Sigma–Aldrich).

Techniques: Activation Assay, Derivative Assay, Transfection, Expressing, Plasmid Preparation, Immunofluorescence

Journal: The Journal of Biological Chemistry

Article Title: The NSs protein of severe fever with thrombocytopenia syndrome virus differentially inhibits the type 1 interferon response among animal species

doi: 10.1016/j.jbc.2023.104819

Figure Lengend Snippet: Interaction of NSs with STAT2. A , NIH3T3 (mouse) cells were transfected with the expression plasmid for HA-tagged NSs and each of the His-tagged STAT2, and then co-IP assays were performed. B , colocalization of NSs with STAT2. NIH3T3 (mouse) cells were transfected with the expression plasmid for HA-tagged NSs and each of the His-tagged STAT2. IFA was also performed with NSs, STAT2, and the nuclei, shown in green , red , and blue , respectively. Scale bar represents 20 μm. C , identification of binding regions in porcine STAT2 to NSs. Schematic representation of the chimeric mutants of human, mouse, and pig STAT2 ( upper ). NIH3T3 (mouse) cells were transfected with the HA-tagged NSs and each of the His-tagged STAT2 chimeras, following which co-IP assays were performed ( lower ). D , the phylogenetic tree of the 101 to 315 region of each STAT2. This phylogenetic tree was constructed using the neighbor-joining method. The bootstrap values are indicated on each node. Representative results of Western blotting assays ( A and C ) and IFA ( B ) are shown. co-IP, coimmunoprecipitation; HA, hemagglutinin; IFA, immunofluorescence assay; NSs, nonstructural protein; STAT, signal transducer and activator of transcription.

Article Snippet: Western blotting was performed as previously described using the following antibodies ( , ): anti-HA (catalog no.: 18850; QED Biosciences, Inc), anti-His (catalog no.: 9F2; Wako), anti-FLAG (catalog no.: F7425; Sigma–Aldrich), anti-STAT2 (catalog no.: D9J7L; Cell Signaling Technology), anti-STAT1 (catalog no.: D19KY; Cell Signaling Technology), anti-STAT2 (phosphor Y690) (catalog no.: GTX50721; GeneTex), anti-STAT1 (phosphor Y701) (catalog no.: D4A7; Cell Signaling Technology), or anti-β-actin (catalog no.: AC-15; Sigma–Aldrich).

Techniques: Transfection, Expressing, Plasmid Preparation, Co-Immunoprecipitation Assay, Binding Assay, Construct, Western Blot, Immunofluorescence

Journal: The Journal of Biological Chemistry

Article Title: The NSs protein of severe fever with thrombocytopenia syndrome virus differentially inhibits the type 1 interferon response among animal species

doi: 10.1016/j.jbc.2023.104819

Figure Lengend Snippet: Function of human, murine, porcine, and chimeric STAT2s in SFTSV infection. SFTSV was inoculated into HEK293T (human) cells transfected with expression plasmid for each STAT2 at MOI 1. After 24 hpi, cells were treated with IFN-αA/D (500 U/ml) for 48 h and then collected culture supernatants. The SFTSV titer in culture supernatants was determined by focus-forming assay ( upper ). The protein expression was detected by Western blotting ( lower ). The assays were independently performed in triplicate. Values are the averages with SDs of data from nine results obtained from three experiments (n = 9). ∗∗ p < 0.01, versus human STAT2. Each exact p value, average, and SD is shown in Table S4 . HEK293T, human embryonic kidney 293T cell line; hpi, hours postinfection; IFN, interferon; MOI, multiplicity of infection; SFTSV, severe fever with thrombocytopenia syndrome virus; STAT, signal transducer and activator of transcription.

Article Snippet: Western blotting was performed as previously described using the following antibodies ( , ): anti-HA (catalog no.: 18850; QED Biosciences, Inc), anti-His (catalog no.: 9F2; Wako), anti-FLAG (catalog no.: F7425; Sigma–Aldrich), anti-STAT2 (catalog no.: D9J7L; Cell Signaling Technology), anti-STAT1 (catalog no.: D19KY; Cell Signaling Technology), anti-STAT2 (phosphor Y690) (catalog no.: GTX50721; GeneTex), anti-STAT1 (phosphor Y701) (catalog no.: D4A7; Cell Signaling Technology), or anti-β-actin (catalog no.: AC-15; Sigma–Aldrich).

Techniques: Infection, Transfection, Expressing, Plasmid Preparation, Focus Forming Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The NSs protein of severe fever with thrombocytopenia syndrome virus differentially inhibits the type 1 interferon response among animal species

doi: 10.1016/j.jbc.2023.104819

Figure Lengend Snippet: Interaction of NSs with STAT1. A , HEK293T (human), NIH3T3 (mouse), CRFK (cat), A72 (dog), Mpf (ferret), and PK15 (pig) cells were transfected with the expression plasmid for HA-tagged NSs, and then co-IP assays were performed. The relative binding rates of NSs to human STAT1 are set at 100%. The assays were independently performed in triplicate. The data represent averages with SDs. The asterisks indicate the nonspecific bands. B , colocalization of NSs with STAT2. The expression plasmid for HA-tagged NSs was transfected to HEK293T (human), NIH3T3 (mouse), CRFK (cat), A72 (dog), Mpf (ferret), and PK15 (pig) cells. IFA was also performed with NSs, STAT1, and the nuclei, shown in green , red , and blue , respectively. Scale bar represents 20 μm. C , interaction of NSs with exogenous STAT1. HEK293T (human), NIH3T3 (mouse), and CRFK (cat) cells were transfected with the expression plasmid for HA-tagged NSs, His-tagged human STAT1 (hSTAT1), murine STAT1 (mSTAT1), feline STAT1 (fSTAT1), and human STAT2 (hSTAT2), and then co-IP assays were performed. D , Interaction activity of NSs to STAT1 and STAT2. The expression plasmid for HA-tagged NSs was transfected into NIH3T3 (mouse) cells with the expression plasmid for His-tagged human STAT1(H) or feline STAT1(F) and FLAG-tagged human STAT2 or feline STAT2, and then co-IP assays were performed. Representative results of Western blotting assays ( A , C , and D ) and IFA ( B ) are shown. CRFK, Crandell–Rees feline kidney; HA, hemagglutinin; HEK293T, human embryonic kidney 293T cell line; IFA, immunofluorescence assay; NSs, nonstructural protein; STAT, signal transducer and activator of transcription.

Article Snippet: Western blotting was performed as previously described using the following antibodies ( , ): anti-HA (catalog no.: 18850; QED Biosciences, Inc), anti-His (catalog no.: 9F2; Wako), anti-FLAG (catalog no.: F7425; Sigma–Aldrich), anti-STAT2 (catalog no.: D9J7L; Cell Signaling Technology), anti-STAT1 (catalog no.: D19KY; Cell Signaling Technology), anti-STAT2 (phosphor Y690) (catalog no.: GTX50721; GeneTex), anti-STAT1 (phosphor Y701) (catalog no.: D4A7; Cell Signaling Technology), or anti-β-actin (catalog no.: AC-15; Sigma–Aldrich).

Techniques: Transfection, Expressing, Plasmid Preparation, Co-Immunoprecipitation Assay, Binding Assay, Activity Assay, Western Blot, Immunofluorescence