Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Bioinspired cardiac-targeted metal-organic framework nanozyme for modulating inflammatory responses in heart failure with preserved ejection fraction

doi: 10.3389/fbioe.2026.1744643

Figure Lengend Snippet: Socs3 expression is elevated in HFpEF hearts and reduced after NanoAM treatment. (A,B) Volcano plots illustrate differential gene expression between groups: HF versus NC (A) , and HFi versus HF (B,C) Venn diagrams display the number of genes with opposite expression patterns in the HFi versus HF comparison relative to the HF versus NC comparison. (D) A Heatmap presents the relative expression levels of the 48 genes across three groups. (E) Interaction network between the 48 genes and insulin resistance (IR)-related genes enriched in , identifying Socs3 as the key overlapping gene.

Article Snippet: Antibodies against IRS1-Ser307 (Proteintech, 85238-1-RR, dilution 1:5000), P-AKT1-T308+AKT2-T309+AKT3-T305 (ABclonal, AP1266, dilution 1:800), SOCS3 (Proteintech, 66797-1-Ig, dilution 1:20,000), and GAPDH (Servicebio, GB15002-100, dilution 1:8000) were used.

Techniques: Expressing, Gene Expression, Comparison

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Bioinspired cardiac-targeted metal-organic framework nanozyme for modulating inflammatory responses in heart failure with preserved ejection fraction

doi: 10.3389/fbioe.2026.1744643

Figure Lengend Snippet: NanoAM regulates the SOCS3-IRS1-AKT2 axis to improve insulin resistance. (A,B) WB analysis of insulin resistance-related proteins in heart tissue (n = 6). (C) The Socs3 mRNA expression levels in heart tissues were determined by qPCR. (D) Immunofluorescence staining of GLUT4 in cardiac tissues. Scale bar, 10 µm. (E) Fluorescence intensity quantification of GLUT4 was shown. (F–I) The Inflammatory factors (IL-6,TNF-α,IL-1β,CRP) mRNA expression levels in heart tissues were determined by qPCR. (J,K) Serum levels of IL-6 and hs-CRP measured by ELISA. Data are showed as mean ± SD and analyzed using one-way ANOVA followed by Tukey’s post hoc test; *p < 0.05, ***p < 0.001.

Article Snippet: Antibodies against IRS1-Ser307 (Proteintech, 85238-1-RR, dilution 1:5000), P-AKT1-T308+AKT2-T309+AKT3-T305 (ABclonal, AP1266, dilution 1:800), SOCS3 (Proteintech, 66797-1-Ig, dilution 1:20,000), and GAPDH (Servicebio, GB15002-100, dilution 1:8000) were used.

Techniques: Expressing, Immunofluorescence, Staining, Fluorescence, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: SOCS1 is an inducible negative regulator of interferon λ (IFN-λ)–induced gene expression in vivo

doi: 10.1074/jbc.M117.788877

Figure Lengend Snippet: IFN-α– but not IFN-λ–induced STAT1 phosphorylation becomes refractory to continuous stimulation. A, liver biopsies from chronic hepatitis C patients (n = 16) were divided into three groups based on their IFNL4 genotype (rs368234815; TT/TT, TT/dG, and dG/dG). Total RNA from biopsies and Huh7 and Huh7 LR cells were prepared. Expression of the IFNLR1 transcript was analyzed by quantitative PCR. Results (mean ± S.D.) are shown as copy numbers per 40 ng of total RNA. B and C, Huh7 LR cells were stimulated with 1000 IU/ml IFN-α or 100 ng/ml IFN-λ1 for the indicated times. B, p-STAT1, STAT1, p-STAT2, STAT2, p-STAT3, STAT3, USP18, SOCS1, SOCS3, and actin were visualized using specific antibodies. Shown are representative blots from two independent experiments. C, transcripts of interferon-stimulated genes (RSAD2, IFI27, and GBP5) were quantified by PCR. Results (mean ± S.D., n = 3) are shown as relative expression to GAPDH. ut, untreated.

Article Snippet: The pCMV6 plasmid containing the SOCS1 gene (RC220847) was purchased from Origene Technologies Inc. (Rockville, MD) and used to overexpress SOCS1.

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: SOCS1 is an inducible negative regulator of interferon λ (IFN-λ)–induced gene expression in vivo

doi: 10.1074/jbc.M117.788877

Figure Lengend Snippet: Overexpression of SOCS1, SOCS3, and USP18 leads to a reduction of IFN-α– and IFN-λ–mediated STAT1 phosphorylation. A, Huh7 LR cells were transiently transfected with control, SOCS1, SOCS3, or USP18 expression plasmids. 24 h later, cells were stimulated with 1000 IU/ml IFN-α or 100 ng/ml IFN-λ1 for 30 min, and p-STAT1, STAT1, SOCS1, SOCS3, USP18, and actin were visualized by immunoblotting. Shown are representative blots from three independent experiments. B, Huh7 LR cells were transfected with SOCS1, SOCS3, or USP18 expression plasmids for 24 h. The mRNA expression levels of SOCS1, SOCS3, and USP18 were analyzed by quantitative PCR and compared with the endogenously induced SOCS1, SOCS3, or USP18 upon IFN-α or IFN-λ1 stimulation at the indicated time points. The results (mean ± S.D., n = 3) are shown as relative expression to GAPDH. Protein levels of SOCS1, SOCS3, and USP18 and actin were visualized using specific antibodies. ox, overexpression; ut, untreated.

Article Snippet: The pCMV6 plasmid containing the SOCS1 gene (RC220847) was purchased from Origene Technologies Inc. (Rockville, MD) and used to overexpress SOCS1.

Techniques: Over Expression, Transfection, Expressing, Western Blot, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: SOCS1 is an inducible negative regulator of interferon λ (IFN-λ)–induced gene expression in vivo

doi: 10.1074/jbc.M117.788877

Figure Lengend Snippet: SOCS1 is a modulator of IFN-λ-signaling. Control, SOCS1−/−, SOCS3−/−, and USP18−/− cells were transfected with pGL3-ISRE-Mx1-Luc and pGL4-CMV-Renilla-Luc plasmids and, 20 h later, stimulated with 100 ng/ml IFN-λ1, 50 ng/ml IFN-λ4, or 1000 IU/ml IFN-α for 4 h, 8 h, and 24 h or left untreated. The firefly luciferase values were normalized to Renilla luciferase, and the results (mean ± S.D., n = 2) are expressed as firefly/Renilla ratio. Unpaired t test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: The pCMV6 plasmid containing the SOCS1 gene (RC220847) was purchased from Origene Technologies Inc. (Rockville, MD) and used to overexpress SOCS1.

Techniques: Transfection, Luciferase

Journal: The Journal of Biological Chemistry

Article Title: SOCS1 is an inducible negative regulator of interferon λ (IFN-λ)–induced gene expression in vivo

doi: 10.1074/jbc.M117.788877

Figure Lengend Snippet: SOCS1 is a modulator of IFN-λ–induced ISGs expression in vitro. Control, SOCS1−/−, SOCS3−/−, and USP18−/− cells were stimulated with 1000 IU/ml IFN-α or 100 ng/ml IFN-λ1 for 4 h, 8 h, and 24 h or left untreated, and the expression levels of RSAD2, GBP5, and IFI27 were analyzed by quantitative PCR. The results (mean ± S.D., n = 2) are shown as relative expression to GAPDH. Unpaired t test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: The pCMV6 plasmid containing the SOCS1 gene (RC220847) was purchased from Origene Technologies Inc. (Rockville, MD) and used to overexpress SOCS1.

Techniques: Expressing, In Vitro, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: SOCS1 is an inducible negative regulator of interferon λ (IFN-λ)–induced gene expression in vivo

doi: 10.1074/jbc.M117.788877

Figure Lengend Snippet: Depletion of Socs-1 increased IFN-λ–induced ISGs expression in vivo. Control and Socs1−/− mice were subcutaneously injected with PBS, 1000 units/g mouse IFN-α, or 50 ng/g mouse IFN-λ2. The liver, the lung, and the gut were collected 4 h and 8 h after injection, and total RNA was prepared. The expression of Rsad2, Oas1, Stat1, Usp18, and Socs1 was measured by quantitative PCR. The results (mean ± S.D.) are shown as relative expression to Rpl19. Three to four animals were used per time point and condition. Unpaired t test with Welch's correction; *, p < 0.05; **, p < 0.01; ***, p < 0.001. ut, untreated.

Article Snippet: The pCMV6 plasmid containing the SOCS1 gene (RC220847) was purchased from Origene Technologies Inc. (Rockville, MD) and used to overexpress SOCS1.

Techniques: Expressing, In Vivo, Injection, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: SOCS1 is an inducible negative regulator of interferon λ (IFN-λ)–induced gene expression in vivo

doi: 10.1074/jbc.M117.788877

Figure Lengend Snippet: Depletion of Usp18 increased IFN-α–induced ISGs expression in vivo. Control and Usp18−/− mice were subcutaneously injected with PBS, 1000 units/g mouse IFN-α, or 50 ng/g mouse IFN-λ2. The liver, the lung, and the gut were collected 4 h and 8 h after injection, and total RNA was prepared. The expression of Rsad2, Oas1, Stat1, Socs1, and Usp18 was measured by quantitative PCR. The results (mean ± S.D.) are shown as relative expression to Rpl19. Three to five animals were used per time point and condition. Unpaired t test with Welch's correction; *, p < 0.05; **, p < 0.01; ***, p < 0.001. ut, untreated.

Article Snippet: The pCMV6 plasmid containing the SOCS1 gene (RC220847) was purchased from Origene Technologies Inc. (Rockville, MD) and used to overexpress SOCS1.

Techniques: Expressing, In Vivo, Injection, Real-time Polymerase Chain Reaction

Journal: Oncology Reports

Article Title: m 6 A methyltransferase METTL3 maintains colon cancer tumorigenicity by suppressing SOCS2 to promote cell proliferation

doi: 10.3892/or.2020.7665

Figure Lengend Snippet: Inhibition of cell proliferation in METTL3-KO SW480 cells. (A) METTL3-KO SW480 cells showed dysregulation of several proliferation-related genes (*P<0.05, compared with the SW480 WT cells). (B) MTS assay results validated the decreased proliferation of METTL3-KO cells (*P<0.05, compared with the SW480 WT cells). (C) METTL3-KO cells showed a reduced proliferation rate, as measured by cell counting (*P<0.05, compared with the SW480 WT cells). (D) METTL3-KO cells showed a lower colony formation rate than control cells at 7 days after seeding. (E) Tumorigenic ability of METTL3-KO SW480 cells was reduced (*P<0.05, compared with the SW480 WT cells). SOCS2, suppressor of cytokine signaling 2; METTL3, methyltransferase like 3; LGR5, leucine-rich repeat-containing G protein-coupled receptor 5; SOX2, SRY-box transcription factor 2; PCNA, proliferating cell nuclear antigen; KO, knockout; SW480 A7 and SW480 A12, METTL3-KO SW480 cells.

Article Snippet: The pCMV6 plasmid containing the full-length SOCS2 sequence (FULL-SOCS2) was purchased from Origene (RC203163).

Techniques: Inhibition, MTS Assay, Cell Counting, Knock-Out

Journal: Oncology Reports

Article Title: m 6 A methyltransferase METTL3 maintains colon cancer tumorigenicity by suppressing SOCS2 to promote cell proliferation

doi: 10.3892/or.2020.7665

Figure Lengend Snippet: SOCS2 is upregulated in colon cancer and downregulated in METTL3-KO SW480 cells. (A) TCGA database analysis revealed a lower expression level of SOCS2 in colon cancer tissues than in the paired normal tissues (*P<0.05). (B) Expression levels of SOCS2 and METTL3 were negatively correlated in colon cancer tissues. (C-E) METTL3-KO upregulated SOCS2 expression at both the (C) mRNA and (D and E) protein levels in SW480 cells (*P<0.05, compared with the SW480 WT cells). SOCS2, suppressor of cytokine signaling 2; METTL3, methyltransferase like 3; KO, knockout; SW480 A7 and SW480 A12, METTL3-KO SW480 cells.

Article Snippet: The pCMV6 plasmid containing the full-length SOCS2 sequence (FULL-SOCS2) was purchased from Origene (RC203163).

Techniques: Expressing, Knock-Out

Journal: Oncology Reports

Article Title: m 6 A methyltransferase METTL3 maintains colon cancer tumorigenicity by suppressing SOCS2 to promote cell proliferation

doi: 10.3892/or.2020.7665

Figure Lengend Snippet: Methylation status of SOCS2 mRNA in SW480 cells. (A) Comparison of the SOCS2 mRNA methylation status between SW480 WT and METTL3-KO SW480 cells via MeRIP-qPCR (*P<0.05, compared with the SW480 WT cells). (B) Dose-dependent increase in SOCS2 mRNA levels in SW480 cells in response to demethylation treatment with DAA (*P<0.05, compared with non-treated group). (C and D) SOCS2 protein expression was upregulated after demethylation treatment with DAA (*P<0.05, compared with the SW380 WT DAA-group). (E) DAA appreciably decreased the m 6 A level in SOCS2 mRNA (*P<0.05, compared with the SW380 WT DAA-group). (F) METTL3-KO increased SOCS2 mRNA stability, showing a decreased mRNA decay rate (*P<0.05, compared with the SW480 WT cells). SOCS2, suppressor of cytokine signaling 2; METTL3, methyltransferase like 3; KO, knockout; SW480 A7 and SW480 A12, METTL3-KO SW480 cells; DAA, 3-deazaadenosine.

Article Snippet: The pCMV6 plasmid containing the full-length SOCS2 sequence (FULL-SOCS2) was purchased from Origene (RC203163).

Techniques: Methylation, Expressing, Knock-Out

Journal: Oncology Reports

Article Title: m 6 A methyltransferase METTL3 maintains colon cancer tumorigenicity by suppressing SOCS2 to promote cell proliferation

doi: 10.3892/or.2020.7665

Figure Lengend Snippet: Modulation of SOCS2 expression alters the proliferation ability of SW480 cells. (A-C) siRNA (si-SOCS2) was used to successfully knock down SOCS2 expression in SW480 cells. Knockdown of SOCS2 had minor impact on METTL3 expression but significantly increased LGR5 expression in SW480 cells (*P<0.05, compared with the si-Ctrl group). (D) Knockdown of SOCS2 enhanced SW480 cell proliferation, as determined by an MTS assay (*P<0.05, compared with the si-Ctrl group). (E-G) Transfection of the pCMV6-SOCS2 plasmid upregulated SOCS2 expression and decreased LGR5 expression in SW480 cells (*P<0.05, compared with the pCMV6-Entry group). SOCS2 overexpression had minor impact on METTL3 expression (P>0.05, compared with the pCMV6-Entry group). (H) SOCS2 overexpression decreased SW480 cell proliferation, as determined by an MTS assay (*P<0.05, compared with the pCMV6-Entry group). SOCS2, suppressor of cytokine signaling 2; METTL3, methyltransferase like 3; LGR5, leucine-rich repeat-containing G protein-coupled receptor 5.

Article Snippet: The pCMV6 plasmid containing the full-length SOCS2 sequence (FULL-SOCS2) was purchased from Origene (RC203163).

Techniques: Expressing, MTS Assay, Transfection, Plasmid Preparation, Over Expression

Journal: Oncology Reports

Article Title: m 6 A methyltransferase METTL3 maintains colon cancer tumorigenicity by suppressing SOCS2 to promote cell proliferation

doi: 10.3892/or.2020.7665

Figure Lengend Snippet: METTL3 modulates LGR5 transcription through m 6 A-mediated changes in SOCS2 expression rather than through direct regulatory mechanisms. (A) TCGA database analysis revealed an induction of LGR5 transcription in colon cancer tissues when compared with normal tissues (**P<0.01, compared with the normal tissues). (B) Expression levels of SOCS2 and LGR5 were negatively correlated in colon cancer tissues. (C) METTL3 knockout (KO) did not obviously impact LGR5 mRNA methylation (as measured by MeRIP-qPCR). (D) METTL3 KO had a minor effect on the LGR5 mRNA decay rate. (E) DAA treatment did not impact LGR5 expression in SW480 cells. (F) Luciferase reporter assay results confirmed that SOCS2 negatively controls LGR5 promoter-driven luciferase activity (*P<0.05, pGL3 basic-LGR5 promoter group vs. FULL SOCS2-LGR5 promoter group). METTL3, methyltransferase like 3; LGR5, leucine-rich repeat-containing G protein-coupled receptor 5; SOCS2, suppressor of cytokine signaling 2; TCGA, The Cancer Genome Atlas; SW480 A7 and SW480 A12, METTL3-KO SW480 cells; DAA, 3-deazaadenosine.

Article Snippet: The pCMV6 plasmid containing the full-length SOCS2 sequence (FULL-SOCS2) was purchased from Origene (RC203163).

Techniques: Expressing, Knock-Out, Methylation, Luciferase, Reporter Assay, Activity Assay

Journal: Oncology Reports

Article Title: m 6 A methyltransferase METTL3 maintains colon cancer tumorigenicity by suppressing SOCS2 to promote cell proliferation

doi: 10.3892/or.2020.7665

Figure Lengend Snippet: Correlations among METTL3, SOCS2 and LGR5 expression levels in CRC. (A-C) Knockdown of METTL3 using siRNA (si-METTL3#1 and si-METTL3#2) significantly upregulated SOCS2 expression but downregulated LGR5 expression in SW480 cells (*P<0.05, compared with the si-Ctrl group). (D and E) Western blotting analysis of 24 paired tissues collected from CRC patients showed high protein levels of METTL3 and LGR5 and a relatively low protein level of SOCS2 in colon cancer (T, colon tumor samples; N, adjacent normal colon tissues; *P<0.05, compared to Normal tissues). (F) Visualization of expression profiles of METTL3, SOCS2 and LGR5 in CRC through the Oncomine database. METTL3 and LGR5 were upregulated in CRC datasets. In contrast, SOCS2 was downregulated in CRC datasets. CRC, colorectal cancer; SOCS2, suppressor of cytokine signaling 2; METTL3, methyltransferase like 3; LGR5, leucine-rich repeat-containing G protein-coupled receptor 5.

Article Snippet: The pCMV6 plasmid containing the full-length SOCS2 sequence (FULL-SOCS2) was purchased from Origene (RC203163).

Techniques: Expressing, Western Blot

Journal: Oncology Reports

Article Title: m 6 A methyltransferase METTL3 maintains colon cancer tumorigenicity by suppressing SOCS2 to promote cell proliferation

doi: 10.3892/or.2020.7665

Figure Lengend Snippet: Association between patient characteristics and gene expression in 24 paired CRC cases.

Article Snippet: The pCMV6 plasmid containing the full-length SOCS2 sequence (FULL-SOCS2) was purchased from Origene (RC203163).

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: The helicase HAGE prevents interferon- α -induced PML expression in ABCB5+ malignant melanoma-initiating cells by promoting the expression of SOCS1

doi: 10.1038/cddis.2014.29

Figure Lengend Snippet: HAGE enhances SOCS1 RNA unwinding and protein expression. ( a ) Schematic representation of the mechanism of action of SOCS1. ( b ) Left panel: IF with antibodies to SOCS1 (green) and HAGE (red) on FM82 control and FM82 shRNA1 and 2 (HAGE knocked down). Right panel: IB with antibodies to HAGE, SOCS1 and actin (loading control) using whole lysates from FM82 and FM55 control and FM82 and FM55 shRNA1 and 2. Scale bar 50 μ m. ( c ) Representative image of immunohistochemistry with antibodies to SOCS1 (green) and HAGE (red) on normal skin and malignant melanoma sections. Scale bar 100 μ m. ( d ) IB with antibodies to HAGE, SOCS1, p-Jak1, p-Tyk2, PML and actin using whole cell extracts from FM82 and FM55 control and FM82 and FM55 shRNA1 and 2 transfected with siRNA control and siRNA for SOCS1 mRNA. ( e ) Transient silencing of SOCS1 in HAGE stable knockdown and control FM82 cells followed by rescue of HAGE expression using a HAGE cDNA expression vector. Actin was measured as a loading control. ( f ) Semi-quantitative real-time PCR of SOCS1 mRNAs isolated from FM82 control and FM82 shRNA1. ( g ) Unwinding of biotinylated SOCS1 N-terminal complimentary RNA sequence-SOCS1 RNA duplexes in the presence of increasing HAGE protein concentrations (lane 1: 0 μ g, lane 2: 0.6 μ g, lane 3: 1.2 μ g). Biotinylated SOCS1 N-terminal complimentary RNA sequence was used as a loading control in lane 4. ( h ) IP with antibody to SOCS1 of biotin alkyle metabolically labelled (30, 60 and 120 min) and unlabelled SOCS1 proteins followed by IB with streptavidin antibody from FM82 control whole cell extracts transfected with SOCS1cDNA and with or without transfected HAGE cDNA

Article Snippet: HAGE or SOCS1 ectopic expression were carried out using pCMV6-DLX5-HAGE (SC126051, OriGene, Rockville, MD, USA) and pCMV6-XL4-SOCS1 (SC111081, Origene).

Techniques: Expressing, Immunohistochemistry, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Isolation, Sequencing, Metabolic Labelling

Journal: Cell Death & Disease

Article Title: The helicase HAGE prevents interferon- α -induced PML expression in ABCB5+ malignant melanoma-initiating cells by promoting the expression of SOCS1

doi: 10.1038/cddis.2014.29

Figure Lengend Snippet: SOCS1 induces ubiquitination of p-Jak1 and p-Tyk2 in FM82 control malignant melanoma cell lines. ( a ) IP with antibody to SOCS1 or rabbit IgG followed by IB with antibodies to SOCS1, p-Jak1 and p-Tyk2 from FM82 control whole cell extracts. ( b and c ) IP with antibody to p-Jak1 or p-Tyk2 and corresponding IgG (rabbit or goat, respectively) followed by IB with antibodies to p-Jak1 or p-Tyk2 and ubiquitin from FM82 control and FM82 shRNA1 whole cell extracts. ( d ) IP with antibody to p-Jak1 followed by whole cell extracts IB with an antibody to ubiquitin from FM82 shRNA1 transfected with HAGE cDNA or SOCS1 cDNA. The protein expression of HAGE and SOCS1 was determined by IB with the corresponding antibodies

Article Snippet: HAGE or SOCS1 ectopic expression were carried out using pCMV6-DLX5-HAGE (SC126051, OriGene, Rockville, MD, USA) and pCMV6-XL4-SOCS1 (SC111081, Origene).

Techniques: Transfection, Expressing

Journal: Cell Death & Disease

Article Title: The helicase HAGE prevents interferon- α -induced PML expression in ABCB5+ malignant melanoma-initiating cells by promoting the expression of SOCS1

doi: 10.1038/cddis.2014.29

Figure Lengend Snippet: HAGE counteracts the anti-proliferative effect of IFN α in ABCB5+ MMICs. ( a , b and c ) IF with antibodies to HAGE (red), ABCB5 (green), SOCS1 (green) and PML (green) on FM82 control, FM82 shRNA1 and 2 melanoma spheres. ( d ) IF and IB with an antibody to PML (or IgG) on FM82 cell lines and corresponding melanoma spheres stably-expressing PML or the empty vector. ( e ) FM82 empty vector and stably expressing PML spheres formation in the presence or absence of cDNA transfected HAGE or SOCS1. ANOVA: *** P =<0.0001. Scale bar 100 μ m (for spheres) and 20 μ m (for cells). ( f ) IF and IB with an antibody to PML on FM82 shRNA/PMLshRNA CTL and FM82shRNA/PMLshRNA 1 and 2. ( g ) Spheres formation assay from FM82shRNA/PMLshRNA CTL and FM82shRNA/PML shRNA 1 and 2. ANOVA: *** P =<0.0001

Article Snippet: HAGE or SOCS1 ectopic expression were carried out using pCMV6-DLX5-HAGE (SC126051, OriGene, Rockville, MD, USA) and pCMV6-XL4-SOCS1 (SC111081, Origene).

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Transfection, shRNA, Tube Formation Assay

Journal: Cell Death & Disease

Article Title: The helicase HAGE prevents interferon- α -induced PML expression in ABCB5+ malignant melanoma-initiating cells by promoting the expression of SOCS1

doi: 10.1038/cddis.2014.29

Figure Lengend Snippet: HAGE counteracts the anti-proliferative effect of IFN α in vivo . ( a , b and c ) FM82 and FM55 controls and FM82 and FM55 shRNA 1 and 2 melanoma sphere formation in the presence or absence of IFN α . Student t -test: ** P =<0.01. ( d ) Summary of the in vivo experimental procedure. ( e ) Measurement of tumour growth in mice injected with either FM82 control cells or FM82 shRNA1 cells and treated or untreated with IFN α ANOVA: ** P =<0.0028; Mann–Whitney U -test: * P =<0.0108 (when comparing FM82 control + PBS and FM82 shRNA + PBS; Mann–Whitney U -test: ** P =<0.0079 (when comparing FM82 control + IFN α and FM82 shRNA + IFN α treated). ( f ) Immunohistochemistry/fluorescence staining with antibodies to HAGE (red), ABCB5 (green), SOCS1 (green) and on sections from FM82 control and FM82 shRNA1 NOD/SCID xenotransplanted tumours and treated or untreated with IFN α . Scale bar 100 μ m

Article Snippet: HAGE or SOCS1 ectopic expression were carried out using pCMV6-DLX5-HAGE (SC126051, OriGene, Rockville, MD, USA) and pCMV6-XL4-SOCS1 (SC111081, Origene).

Techniques: In Vivo, shRNA, Injection, MANN-WHITNEY, Immunohistochemistry, Fluorescence, Staining

Journal: The Journal of Experimental Medicine

Article Title: NOS1-derived nitric oxide promotes NF-κB transcriptional activity through inhibition of suppressor of cytokine signaling-1

doi: 10.1084/jem.20140654

Figure Lengend Snippet: NOS1-derived NO mediates S-nitrosation of SOCS1 and prevents SOCS1-mediated proteasomal degradation of p65 . (A) Immunoblot analysis of p65 and SOCS1 in WT, NOS1 −/− , NOS2 −/− , and NOS3 −/− BMDM treated with LPS (250 ng/ml) for the indicated times. (B) SOCS1 S-nitrosation was detected using the biotin switch method on protein from WT and NOS1 −/− BMDMs pretreated with MG132 (50 µM) for 1 h before treatment with LPS (250 ng/ml) for the indicated times, followed by immunoblotting for SOCS1. (C) NOS2 −/− and NOS3 −/− BMDMs analyzed by biotin switch method, as in B. (D) Co-immunoprecipitation of SOCS1 and p65 in WT and NOS1 −/− BMDMs, pretreated with MG132 (50 µM, 1 h) before LPS (250 ng/ml) for the indicated time intervals. (E) Immunoblot analysis of p65 and SOCS1 total protein levels in NOS1 −/− BMDM treated with DEANO (NO donor, 5 µM) and LPS (250 ng/ml). All blots shown are representative of at least two (C and D) or three (A, B, and E) independent experiments.

Article Snippet: Cells were transiently transfected with pCMV6-AC-GFP Vector (OriGene), which expresses SOCS1 (mouse; available from GenBank under accession no. NM_009896.2 ) driven by the constitutive CMV promoter, using Lipofectamine 2000 reagent according to the manufacturer’s protocol.

Techniques: Derivative Assay, Western Blot, Immunoprecipitation

Journal: The Journal of Experimental Medicine

Article Title: NOS1-derived nitric oxide promotes NF-κB transcriptional activity through inhibition of suppressor of cytokine signaling-1

doi: 10.1084/jem.20140654

Figure Lengend Snippet: Molecular modeling and functional testing demonstrate that Cys147 and Cys179 of SOCS1 are the targets for S-nitrosation. (A) Protein domain structure of SOCS1 illustrating the positions of all 5 candidate cysteine residues. (B) Molecular modeling was performed to predict the accessibility of cysteine residues of SOCS1 (represented as colored stick atomic models) to the likely transnitrosation donor, GSNO (represented as ball and stick atomic models). The shorter distances between the cysteine sulfur atom and the nitrogen of GSNO are predicted to permit S-nitrosation for Cys147 (4 Å, middle) and Cys179 (6 Å, right), whereas Cys43 (13 Å, left), Cys112 (11 Å, not depicted), and Cys78 (11 Å, not depicted) are predicted to be too far apart to permit the reaction. (C) Representative immunoblots of GFP-tagged constructs of SOCS1 or point mutations of each cysteine (C147S, C179S, C112S, C78S, and C43S) were transfected into HEK293 cells, which were then treated with a 15-min pulse of 20 ng/ml IL-1β. Some of the cells were pretreated with 50 µM proteasome inhibitor for 1 h (MG132 +) and some were treated with 10 µM DEANO for 1 h after IL-1β (NO donor +). Stability of SOCS1 protein levels was determined by immunoblot and (D) quantified relative to β-actin from three independent experiments (mean ± SEM). (E) The capacity of SOCS1 mutants C147S, and C179S for S-nitrosation was compared with WT SOCS1 in transiently transfected HEK293 cells using the biotin switch method. Immunoprecipitates of GFP-SOCS1 variants were assayed for biotin (S-nitrosation), and lysates were immunoblotted for total SOCS1 as control. Data are representative of two independent experiments.

Article Snippet: Cells were transiently transfected with pCMV6-AC-GFP Vector (OriGene), which expresses SOCS1 (mouse; available from GenBank under accession no. NM_009896.2 ) driven by the constitutive CMV promoter, using Lipofectamine 2000 reagent according to the manufacturer’s protocol.

Techniques: Functional Assay, Western Blot, Construct, Transfection