Journal: Theranostics

Article Title: H3K27 acetylation activated-COL6A1 promotes osteosarcoma lung metastasis by repressing STAT1 and activating pulmonary cancer-associated fibroblasts

doi: 10.7150/thno.51245

Figure Lengend Snippet: COL6A1 promotes OS invasion and migration via suppressing STAT1 expression and activation. A. Scatter plot of top 20 KEGG pathways enrichment of DEGs after COL6A1 transfection. Rich factor is the ratio of the DEG number to the background number in a certain pathway. The size of the dots represents the number of genes, and the color of the dots represents the range of the q-value. B. STAT1 and STAT3 mRNA levels were determined in OS cell with COL6A1 or siCOL6A1 transfection by qRT-PCR. C. Total and phosphorylated STAT1 and STAT3 protein expression levels were analyzed in COL6A1 overexpression and knockdown in OS cells by western blot. D. Nuclear and cytoplasmic STAT1 expression was analyzed in COL6A1 overexpression or control OS cells. E. STAT1 transcription luciferase reporter constructs were transiently transfected into the indicated cells, and luciferase activity was analyzed after 48 hours. F. Biotin pull-down assay with a STAT1 probe was used to determine its DNA binding after transfecting COL6A1. G. STAT1 overexpression decreased the migratory ability of COL6A1 overexpression OS cells. The migratory ability of OS cells was detected upon the indicated treatment (right panel). H . The expression of STAT1 in OS tissues was detected by western blot. I. Expression of STAT1 and COL6A1 in OS tissues was detected by immunohistochemistry and COL6A1 inversely correlated with STAT1 expression in human OS tissues (Scale bars: 100 µm). J. Overexpression of STAT1 decreased the rate of lung metastasis after tail-vein injection indicated cells (n = 6 each group). Representative photographs of H&E staining in lung metastases tissues from mice orthotopically inoculated with indicated treated cells (Scale bars: 400 µm). Data represent the mean ± SD of 3 separate determinations. * p < 0.05, ** p < 0.01, *** p < 0.001 by Student's t test.

Article Snippet: GFP-tagged wild type STAT1 (wt), GFP-tagged STAT1 (Y701A), and GFP-tagged STAT1 (S727A) were purchased from Addgene (Watertown, MA, USA).

Techniques: Migration, Expressing, Activation Assay, Transfection, Quantitative RT-PCR, Over Expression, Western Blot, Luciferase, Construct, Activity Assay, Pull Down Assay, Binding Assay, Immunohistochemistry, Injection, Staining

Journal: Theranostics

Article Title: H3K27 acetylation activated-COL6A1 promotes osteosarcoma lung metastasis by repressing STAT1 and activating pulmonary cancer-associated fibroblasts

doi: 10.7150/thno.51245

Figure Lengend Snippet: COL6A1 interacted with E3 ligase SOCS5 to promote STAT1 degradation. A. Co-localization of COL6A1 and STAT1 in ESCC cells were examined by using confocal microscopy (scale bar, 10 µm). B. The interaction of GFP-STAT1 and Flag-COL6A1 wide type, VWFA1, VWFA2, VWFA3 domains deletion mutations were detected by co-immunoprecipitation in 293T cells. C. COL6A1 was transfected into the OS cells in the presence of cycloheximide (CHX, 200 µg/mL) for indicated times. Cell lysates were immunoblotted by antibodies as indicated. The data were quantified using Image J software. D. STAT1 ubiquitination was detected by immunoprecipitation with anti-STAT1 antibody and immunoblotting with an anti-Ub antibody. E. The interaction of STAT1, COL6A1 and SOCS5 was detected by co-immunoprecipitation in U2OS. F. SOCS5 decreased STAT1 protein. U2OS cells were transfected with Flag-SOCS5 or Flag-SOCS5 R406K as well as control or COL6A1 transfection. The protein expression level of STAT1 was assayed by western blot. G . The cells expressing wide type SOCS5 were treated with CHX. The protein levels of STAT1 and SOCS5 were analyzed by western blot. H . Knockdown SOCS5 increased STAT1 protein. U2OS cells were transfected with si-SOCS5 as well as control or COL6A1 transfection. I . U2OS cells were transfected with control or SOCS5 siRNAs treated with CHX (200 µg/mL), the protein levels of STAT1 and SOCS5 were analyzed by western blot. J. SOCS5 ubiquitylates STAT1. U2OS cells were transfected with indicated plasmids or siRNA for 48 h. Cell lysates were immunoprecipitated with anti-GFP and analyzed by immunoblotting with indicated antibodies. K . U2OS cells were transfected with indicated plasmids. lysates were immunoprecipitated with anti-GFP, and western blots were performed to analyze the presence of indicated proteins and levels of ubiquitination. Data represent the mean ± SD of 3 separate determinations. * p < 0.05, ** p < 0.01, *** p < 0.001 by Student's t test.

Article Snippet: GFP-tagged wild type STAT1 (wt), GFP-tagged STAT1 (Y701A), and GFP-tagged STAT1 (S727A) were purchased from Addgene (Watertown, MA, USA).

Techniques: Confocal Microscopy, Immunoprecipitation, Transfection, Software, Western Blot, Expressing

Journal: Theranostics

Article Title: H3K27 acetylation activated-COL6A1 promotes osteosarcoma lung metastasis by repressing STAT1 and activating pulmonary cancer-associated fibroblasts

doi: 10.7150/thno.51245

Figure Lengend Snippet: Activated fibroblasts promote OS metastasis by secreting TGF-βA. The cell invasion ability was detected by transwell assay after TGF-β treatment. B. COL6A1 expression was detected by western blot, confocal microscope and qRT-PCR in OS cells exposed to mock vehicle or TGF-β (0-10 ng/mL) for 12 h. C . COL6A1 protein and mRNA expression in U2OS cells transfected with siRNAs targeting SMAD2 exposed to mock vehicle or TGF-β (5 ng/mL) for 12 h. D. COL6A1-knockdown Saos-2 cells were treated with TGF-β, western blot were performed to detect the expression of COL6A1. E . COL6A1-knockdown Saos-2 cells were treated with TGF-β and transwell assay were performed to detect the migration ability of OS cells. F. The expressions of E-cadherin, N-cadherin, β-catenin and vimentin in OS cells with COL6A1 or si-COL6A1 transfection by western blot, qRT-PCR and confocal microscope (Scale bars: 50 µm). H. COL6A1-knockdown U2OS cells were treated with TGF-β, western blots and qRT-PCR were performed on cell lysates with the indicated antibodies . The protein and mRNA expression of TGF-β was detected in COL6A1 overexpressed OS cells. I. The concentration of TGF-β in OS patients' serum was detected by ELISA (non-Meta: non-metastasis OS patients; Meta: lung metastasis OS patients; H.C.: healthy control). J. Schematic diagram summarizing how COL6A1 promotes OS metastasis via suppressing STAT1 expression and activating CAFs. Exo, exosome. Data represent the mean ± SD of 3 separate determinations. * p < 0.05, ** p < 0.01, *** p < 0.001 by Student's t test.

Article Snippet: GFP-tagged wild type STAT1 (wt), GFP-tagged STAT1 (Y701A), and GFP-tagged STAT1 (S727A) were purchased from Addgene (Watertown, MA, USA).

Techniques: Transwell Assay, Expressing, Western Blot, Microscopy, Quantitative RT-PCR, Transfection, Migration, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Transcriptional control of pancreatic cancer immunosuppression by metabolic enzyme CD73 in a tumor-autonomous and -autocrine manner

doi: 10.1038/s41467-023-38578-3

Figure Lengend Snippet: a Top upregulated pathways identified from transcriptomic data of KPC cells with and without CD73 overexpression. b , c p38 inhibition reduces the expression of CCL5 at the transcriptional level. mRNA levels of CCL5 in ADO-preincubated KPC cells ( b ) and BXCP-3 cells ( c ) treated with MK-2006, temuterkib, ERK5-IN-1, SB20350, or JNK inhibitor IX ( n = 4). d CD73 regulates the phosphorylation of p38. Immunoblot analysis of CD73, p-p38, and p38 in CD73 KO/OE KPC cells. e Regulation of p38 phosphorylation by CD73 via ADO–Adora2a signaling. Immunoblot analysis of CD73, p-p38, and p38 in CD73 KO/OE cells treated with or without KW6002. f ADO–Adora2a signaling regulates CCL5 expression through p38 activation. Immunoblot analysis of CCL5 in KPC and BXPC-3 cells treated with or without adenosine and SB203580. g Correlation analysis between classical downstream transcription factors and CCL5 based on the TCGA database. h CD73 regulates the phosphorylation of STAT1. Immunoblot analysis of CD73, p-STAT1, and STAT1 in CD73 KO/OE KPC cells. i Direct binding of STAT1 to the promoter and enhancer of CCL5. ChIP assay of the binding between STAT1 and three predicted regions in the CCL5 gene promoter in both SW1990 and BXCP-3 cells. j , k Regulation of the p38–STAT1 axis by CD73 via ADO–Adora2a signaling. Immunoblot analysis of CD73, p-p38, p38, p-STAT1, and STAT1 in CD73 WT/KO BXPC-3 ( j ) and KPC ( k ) cells treated with or without adenosine. l Immunoblot analysis of p-p38, p38, p-STAT1, and STAT1 in BXPC-3 and KPC cells treated with or without adenosine and KW6002. m Abolition of CCL5 upregulation induced by CD73 overexpression through STAT1 depletion. Immunoblot analysis of CD73, STAT1, and CCL5 in WT and CD73 KO KPC cells and BXPC-3 cells with or without STAT1 depletion. All data are representative of three independently performed experiments. Results represent means ± SD of one representative experiment in ( b , c ). * P < 0.05, ** P < 0.01, *** P < 0.001 using a two-tailed t -test; ns: not significant. The Spearman correlations and P -values by Spearman’s test are indicated in ( g ). The exact P -values are shown in the Source Data. Source data are provided as a Source Data file.

Article Snippet: Similarly, to generate STAT1 KD cells, KPC and BXPC-3 cells were transfected with human STAT1 double nickase plasmid (sc-400086-NIC, Santa Cruz) and mouse STAT1 double nickase plasmid (sc-423174-NIC, Santa Cruz) and then sorted by GFP fluorescence.

Techniques: Over Expression, Inhibition, Expressing, Western Blot, Activation Assay, Binding Assay, Two Tailed Test

Journal: bioRxiv

Article Title: STAT1 dissociates adipose tissue inflammation from insulin sensitivity in obesity

doi: 10.1101/2020.04.10.036053

Figure Lengend Snippet: (A) Relative mRNA expression of Stat1 in lean (grey) or high fat diet-induced obese (DIO) (red) wild-type mice (n=4/group). *p<0.05, data are represented as mean +/− SEM. (B) Relative STAT1 expression was measured in human subcutaneous adipose tissue biopsied from subjects with prediabetes (red; n=11) compared to those with normal glucose tolerance (NGT, grey; n=18) #p<0.09). Human subcutaneous preadipocytes were differentiated for 8 days and then transfected with STAT1 or control vector (pcDH). *p<0.05, data are represented as mean +/− SEM. (C) To confirm STAT1 expression, immunoblotting of total STAT1 and FLAG was performed, along with markers of mature adipocytes and mitochondrial proteins. (D) Respiration (as oxygen consumption rate, OCR) was measured in human adipocytes expressing control vector (pcDH; grey line) or STAT1 (red line) over time with the addition of oligomycin (α), carbonyl cyanide- 4 -(trifluoromethoxy) phenylhydrazone (FCCP) (β), and antimycin-A/rotenone (γ) (n=5). *p<0.05 indicates changes in maximal respiration.

Article Snippet: FLAG- STAT1 (Addgene #71454) or vector control lentiviral particles were prepared and introduced into human adipocytes for 48h before experiments.

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

Journal: bioRxiv

Article Title: STAT1 dissociates adipose tissue inflammation from insulin sensitivity in obesity

doi: 10.1101/2020.04.10.036053

Figure Lengend Snippet: Human subcutaneous preadipocytes were differentiated for 10 days and then transfected with STAT1 siRNA or scramble RNA (scRNA; control) for 48h. After transfection, cells were treated +/− 100 ng/ml IFN γ for 24h. (A) Relative mRNA expression of STAT1 , IRF1 , UCP1 , and ADIPOQ (n=9). grey – scRNA, red – STAT1 siRNA; *p<0.05 vs scRNA, #p<0.05 vs -IFN γ vehicle treated; data are represented as mean +/− SEM. (B) Oxygen consumption rate (OCR) in differentiated human adipocytes after exposure to IFN γ with addition of oligomycin (α), carbonyl cyanide- 4 -(trifluoromethoxy) phenylhydrazone (FCCP) (β), and antimycin-A/rotenone (γ). (black – scRNA vehicle, green – scRNA +IFN γ , blue – STAT1 siRNA vehicle, red – STAT1 siRNA + IFN γ ) (n=12); *p<0.06 vs scRNA, #p<0.06 vs -IFN γ vehicle treated for maximal respiration. 3T3-L1 cells were transfected with Cas9 and one of three Stat1 single guide RNAs (g1, g2, g3) or a non-mammalian targeting control (ntCR1) guide RNA. (C) Immunoblots of total lysates from ntCR1 or g3 Stat1 (gSTAT1) cells +/− differentiation for 10 days. (D) Relative mRNA expression of Stat1 , Ppar γ 2 , AdipoQ , and Pgc1a from ntCR1 and g3 Stat1 cells +/− differentiation for 10 days (n=3). (E) Differentiated ntCR1 and gSTAT1 cells were treated +/− 100 ng/ml IFN γ for 24h and then harvested for quantification of relative mRNA for inflammatory ( Stat1 , Irf1 ), fat cell identity ( Ppar γ 2 , AdipoQ ), and lipid metabolism genes ( Pgc1a , Acly , Aspa ) (n=3). grey – ntCR1; red – gSTAT1; *p<0.05 vs ntCR1, #p<0.05 vs vehicle; data are represented as mean +/− SEM.

Article Snippet: FLAG- STAT1 (Addgene #71454) or vector control lentiviral particles were prepared and introduced into human adipocytes for 48h before experiments.

Techniques: Transfection, Control, Expressing, Western Blot

Journal: bioRxiv

Article Title: STAT1 dissociates adipose tissue inflammation from insulin sensitivity in obesity

doi: 10.1101/2020.04.10.036053

Figure Lengend Snippet: (A) Immunoblots show STAT1 knockdown in iWAT and eWAT, but not liver, of fat specific (ADIPOQ-Cre) STAT1 fKO compared to STAT1 fl/fl littermate controls. (B) Immunoblots show unaltered STAT2 and STAT3 expression in iWAT and eWAT of STAT1 fKO mice. (C) Relative Stat1 expression in the iWAT and eWAT of STAT1 fl/fl and STAT1 fKO mice (n=7-8/group). (D) Body mass and (E) composition (% body mass) for STAT1 fl/fl and STAT1 fKO mice on HFD for 18 weeks (n=9-11/group). (F-H) Insulin (ITT; n=12-13/group) and glucose (GTT; n=8/group) tolerance tests with corresponding overnight fasting serum insulin (n=8/group) in STAT1 fl/fl and STAT1 fKO mice on HFD. (I) eWAT and (J) iWAT staining for macrophages (Mac3; brown) and (K) relative mRNA expression of inflammatory genes. (L) Adipocyte cell size distribution (% total cells) tabulated across four 20x fields of view per mouse fat depot (n=5-6/group) grey – STAT1 fl/fl ; red – STAT1 fKO *p<0.05, data are represented as mean +/− SEM.

Article Snippet: FLAG- STAT1 (Addgene #71454) or vector control lentiviral particles were prepared and introduced into human adipocytes for 48h before experiments.

Techniques: Western Blot, Knockdown, Expressing, Staining

Journal: bioRxiv

Article Title: STAT1 dissociates adipose tissue inflammation from insulin sensitivity in obesity

doi: 10.1101/2020.04.10.036053

Figure Lengend Snippet: (A) RNA-Seq coupled with (B) GSEA identified gene signatures altered by STAT1 knockout in the eWAT and iWAT of obese mice. Relative mRNA expression of key genes that validate the (C) anti-inflammatory and (D) metabolic gene signatures of STAT1 deletion in iWAT. (E) Heatmap representing hierarchical clustering of differential metabolites in iWAT between obese STAT1 fl/fl and STAT1 fKO mouse models (n=5/group; FDR<0.25) and (F) lean or diet-induce obese wild-type mice (n=4-6/group) were assessed using mass spectrometry. (G) Metabolomics analysis of iWAT establish HFD feeding in WT mice reduced (red) tricarboxylic acid (TCA) cycle metabolites that become rescued (green) in STAT1 fKO mice. G6P/F6P – glucose/fructose-6-phosphate; GBP/FBP – glucose/fructose-1,6-bisphosphate; 3PG/2PG – 3-/2-phosphoglycerate; G3P – glyceraldehyde 3-phosphate; PEP – phosphoenolpyruvate; Ac-CoA – acetyl-CoA; -KG – alpha-ketoglutarate. grey – STAT1 fl/fl ; red – STAT1 fKO ; *p<0.05, data are represented as mean +/− SEM.

Article Snippet: FLAG- STAT1 (Addgene #71454) or vector control lentiviral particles were prepared and introduced into human adipocytes for 48h before experiments.

Techniques: RNA Sequencing, Knock-Out, Expressing, Mass Spectrometry

Journal: bioRxiv

Article Title: STAT1 dissociates adipose tissue inflammation from insulin sensitivity in obesity

doi: 10.1101/2020.04.10.036053

Figure Lengend Snippet: (A) Body weight gain (% initial; n=4-5/group) and (B) body composition (MRI; n=9/group) measured after 12 weeks of HFD. Insulin sensitivity was determined by (C) insulin tolerance tests in obese IFN γ gR1+/+ and IFN γ gR1 −/- mice (n=9 mice/group, *p<0.05). (D) Serum insulin levels and (E) HOMA-IR were assessed in fasted mice (n=9 mice/group, *p<0.05). (F) Serum leptin levels were assessed in obese mice fasted 4 h (n=9 mice/group *p<0.05). (G) iWAT H/E and (H) adipocyte cell size distribution (% total cells) tabulated across four 20x fields of view per mouse fat depot (n=4-5/group) grey – IFN γ gR1+/+ ; red – IFN γ gR1 −/- . (I) IFN γ -STAT1 inflammation and metabolism genes from iWAT of IFN γ gR1+/+ and IFN γ gR1 −/- mice on HFD (n=13-14/group). (J) Metabolite levels in iWAT of obese IFN γ gR1+/+ (grey) and IFN γ gR1 −/- (red) mice (n=4-5/group) were assessed using mass spectrometry. *p<0.05, data are represented as mean +/− SEM. Red metabolites decreased by HFD in WT mice; IFN γ gR deletion rescued (green) and increased (blue) metabolites. (K) Validation of IFN γ gR1 deletion and impaired STAT1 signaling by Western blot analysis of total cell lysates from IFN γ gR1+/+ and IFN γ gR1 −/- SVF-derived adipocytes after 24 h exposure to IFN γ . Relative mRNA expression of (L) Stat1 , Oas1 , AdipoQ , Ucp1 from IFN γ gR1+/+ (grey) and IFN γ gR1 −/- (red) adipocytes after exposure to IFN γ (n=3, *p<0.05 vs IFN γ gR1+/+ , #p<0.05 vs vehicle). (M) Respiration (as oxygen consumption rate, OCR) was measured in IFN γ gR1+/+ and IFN γ gR1 −/- adipocytes after IFN γ treatment and during oligomycin (α), Carbonyl cyanide- 4 -(trifluoromethoxy)phenylhydrazone (FCCP) (β), and antimycin-A/Rotenone (γ) (n=5, *p<0.05 vs IFN γ gR1+/+ , #p<0.05 vs vehicle) additions. (black – IFN γ gR1+/+ vehicle, blue – IFN γ gR1+/+ +IFN γ , green – IFN γ gR1 −/- vehicle, red – IFN γ gR1 −/- +IFN γ ). Data are represented as mean +/− SEM.

Article Snippet: FLAG- STAT1 (Addgene #71454) or vector control lentiviral particles were prepared and introduced into human adipocytes for 48h before experiments.

Techniques: Mass Spectrometry, Biomarker Discovery, Western Blot, Derivative Assay, Expressing

Journal: The Journal of Clinical Investigation

Article Title: Isocitrate dehydrogenase mutations suppress STAT1 and CD8 + T cell accumulation in gliomas

doi: 10.1172/JCI90644

Figure Lengend Snippet: (A) Western blotting was performed on GL261-WT and GL261-MUT cell lysates in the presence or absence of 1 μM IDH-C35 and 100 ng/ml recombinant murine IFN-γ. (B) Quantification of Western blot bands by ImageJ. Data represent the mean ± SD of band density/β-actin of 2 to 4 experiments. (C) Western blotting was performed on cell lines treated with 3 mM 2HG. Data shown represent GL261 cells treated with 2HG for 1, 3, or 5 days and NHA and SB28 cells treated with 2HG for 5 days. (D) ImageJ quantification of Western blot bands from C. Data represent the mean ± SD of band density/β-actin band density from 3 independent experiments. (E) ImageJ quantification of Western blot band densities of STAT1, p-STAT1, and IRF1, normalized to β-actin levels for NHA and SB28 cells treated with 2HG. Data are representative of 2 independent experiments with similar results.

Article Snippet: Data obtained by 2-tailed unpaired t test. ( C ) RT-PCR analysis of Stat1 , Cxcl9 , and Cxcl10 expression in GL261 WT cells stimulated with 10 ng/ml recombinant murine IFN-γ for 12 hours, 36 hours after transfection with 1 μg plasmid encoding either scrambled shRNA (OriGene Technologies; TR30013) or 4 shRNAs targeting STAT1 (OriGene Technologies; TG502153). ( D ) CXCL10 ELISA was performed using supernatant from the cells described in C . Data shown in C and D represent the mean ± SD of 3 biologic replicate samples and are representative of 2 independent experiments; *** P < 0.0001. sh-Ctrl, sh-control.

Techniques: Western Blot, Recombinant

Journal: The Journal of Clinical Investigation

Article Title: Isocitrate dehydrogenase mutations suppress STAT1 and CD8 + T cell accumulation in gliomas

doi: 10.1172/JCI90644

Figure Lengend Snippet: (A) Representative images showing STAT1 staining on IDH-WT and IDH-MUT WHO grade III LGG samples. Scale bars: 80 μm and 8 μm. (B) Quantification of STAT1+ cells per area of tumor (mm2) on IDH-WT (56 sections from 9 cases) and IDH-MUT (23 sections from 11 cases) tumors. Data represent the mean ± SD. Data obtained by 2-tailed unpaired t test. (C) RT-PCR analysis of Stat1, Cxcl9, and Cxcl10 expression in GL261WT cells stimulated with 10 ng/ml recombinant murine IFN-γ for 12 hours, 36 hours after transfection with 1 μg plasmid encoding either scrambled shRNA (OriGene Technologies; TR30013) or 4 shRNAs targeting STAT1 (OriGene Technologies; TG502153). (D) CXCL10 ELISA was performed using supernatant from the cells described in C. Data shown in C and D represent the mean ± SD of 3 biologic replicate samples and are representative of 2 independent experiments; ***P < 0.0001. sh-Ctrl, sh-control.

Article Snippet: Data obtained by 2-tailed unpaired t test. ( C ) RT-PCR analysis of Stat1 , Cxcl9 , and Cxcl10 expression in GL261 WT cells stimulated with 10 ng/ml recombinant murine IFN-γ for 12 hours, 36 hours after transfection with 1 μg plasmid encoding either scrambled shRNA (OriGene Technologies; TR30013) or 4 shRNAs targeting STAT1 (OriGene Technologies; TG502153). ( D ) CXCL10 ELISA was performed using supernatant from the cells described in C . Data shown in C and D represent the mean ± SD of 3 biologic replicate samples and are representative of 2 independent experiments; *** P < 0.0001. sh-Ctrl, sh-control.

Techniques: Staining, Reverse Transcription Polymerase Chain Reaction, Expressing, Recombinant, Transfection, Plasmid Preparation, shRNA, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Interferon regulatory factor 3 is a key regulation factor for inducing the expression of SAMHD1 in antiviral innate immunity

doi: 10.1038/srep29665

Figure Lengend Snippet: ( A ) MARC-145 cells and PAMs were infected or mock infected with PRRSV or NDV at an MOI of 1 for 16 h. The levels of SAMHD1 expression, phosphorylation of IRF3, and STAT1, were analyzed using Western blotting. ( B ) HEK293 cells and MARC-145 cells were transfected with psiRNA vector expressing shRNA targeting STAT1 gene for 36 h, then the cells were cultured in selective medium 50–150 μg/mL Zeocin (Life technologies) for 3 days until cell foci were identified. The cells were treated with IFN-α for 12 h. STAT1 and SAMHD1 expression were analyzed using Western blotting. ( C ) HEK293 and MARC-145 cells transfected with STAT1 WT or STAT1 Y701F plasmids were analyzed for SAMHD1 expression at 48 h post-transfection using western blotting. ( D ) THP-1 cells were either non-differentiated or differentiated overnight with 50 ng/ml of PMA, and then treated with 1,000 U/ml human IFN-α for 0–6 h. Nuclear proteins were extracted and the nuclear translocation of STAT1, IRF3, and SAMHD1 expression were detected using Western blotting. PCNA was used as a protein loading control. Expression levels of SAMHD1 compared to β-actin or PCNA are shown. ( E ) THP-1 cells were mock treated or treated with 1,000 U/mL IFN-α for the indicated times. Quantitative RT-PCR was performed using SAMHD1 specific primers and all data was normalized to β-actin (NS, not significant: p > 0.05). Uncropped images of blots are shown in .

Article Snippet: STAT1 WT and STAT1 Y701F plasmids were purchased from Addgene.

Techniques: Infection, Expressing, Phospho-proteomics, Western Blot, Transfection, Plasmid Preparation, shRNA, Cell Culture, Translocation Assay, Control, Quantitative RT-PCR