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: Advanced Science

Article Title: The Crosstalk Between CRL5 and APC/C E3 Ligases Regulates Metastasis and Chemosensitivity of Cancer Cells

doi: 10.1002/advs.202512652

Figure Lengend Snippet: APC11 depletion promotes CUL5 neddylation but stabilizes integrin β1. a) Immunoblot of indicated proteins in PLC/PRF/5, Hep3B, and PANC‐1 cells transfected with siRNA targeting APC11 or with a scrambled control siRNA. b) Co‐IP of transfected FLAG‐CUL5 and endogenous CAND1, COPS5, UBE2F or SAG in HEK293 cells transfected with indicated siRNA oligos. c) Co‐IP of endogenous CUL5 with UBE2F or APC11 in A549 cells transfected with indicated siRNA oligos. d) Immunoblot of CUL5 in A549 and PLC/PRF/5 cells transfected with indicated siRNA oligos. e) The stability of SOCS3 and integrin β1 in A549 and MIA PaCa‐2 cells upon APC11 knockdown. A549 and MIA PaCa‐2 cells were transfected with indicated siRNA oligos for 72 h and then treated with 100 µg mL −1 CHX for the indicated time periods, followed by IB analysis. Densitometry quantifications were performed with ImageJ, and the decay curves are shown (right). Data are presented as mean ± SEM, n = 3. For statistical analysis, significances were determined by Student's t ‐test. * p < 0.05, ** p < 0.01, *** p < 0.001. f) Diagram illustrating how APC11 depletion promotes CUL5 neddylation and leads to integrin β1 accumulation.

Article Snippet: [ ] The following antibodies were used: ACTIN (Sigma–Aldrich, A5441), FLAG (Sigma–Aldrich, F1804), FLAG (Sigma–Aldrich, F7425), GFP (Santa Cruz, sc‐9996), MYC (Santa Cruz, sc‐789), APC2 (Cell Signaling Technology, 12301), APC8 (Cell Signaling Technology, 15100), APC10 (Cell Signaling Technology, 14807), APC11 (Cell Signaling Technology, 14090), APC11 (Santa Cruz, sc‐517142), SAG (Proteintech, 11905‐1‐AP), RBX1 (Proteintech, 14895‐1‐AP), CUL1 (Santa Cruz, sc‐11384), CUL5 (Abcam, ab184177), CUL5 (Santa Cruz, sc‐373822), CAND1 (Abcam, ab183748), integrin β1 (Cell Signaling Technology, 34 971), p‐SRC (Y416) (Cell Signaling Technology, 6943), ATM (Cell Signaling Technology, 2873), Cyclin B1 (Cell Signaling Technology, 4138), RPB1 (Cell Signaling Technology, 14958), EGFR (Cell Signaling Technology, 4267), DEPTOR (Cell Signaling Technology, 11 816), NOXA (Millipore, OP180), UBE2F (Proteintech, 17056‐1‐AP), COPS5 (Cell Signaling Technology, 6895), SOCS3 (Proteintech, 14025‐1‐AP), LC3 (Cell Signaling Technology, 2775), securin (Cell Signaling Technology, 13445), ITCH (Proteintech, 20920‐1‐AP), Cyclin E1 (Cell Signaling Technology, 20808), p‐H3 (Cell Signaling Technology, 3377), and p‐Aurora A/B/C (Cell Signaling Technology, 2914), Aurora A (Cell Signaling Technology, 14 475), Aurora B (Abcam, ab2254), and NEDD8 (Abcam, ab81264).

Techniques: Western Blot, Transfection, Control, Co-Immunoprecipitation Assay, Knockdown

Journal: mBio

Article Title: DDX4 enhances antiviral activity of type I interferon by disrupting interaction of USP7/SOCS1 and promoting degradation of SOCS1

doi: 10.1128/mbio.03213-23

Figure Lengend Snippet: DDX4 inhibits the interaction of USP7 with SOCS1. ( A ) Potential of interaction protein of DDX4 obtained from the PINA database. ( B ) Immunoprecipitation analysis of the interaction between HA-USP7 and Myc-DDX4 by immunoprecipitating Myc-DDX4 in HEK293T cells cotransfected with HA-USP7 and Myc-DDX4. ( C ) Immunoprecipitation analysis of the interaction between HA-USP7 and Myc-DDX4 by immunoprecipitating HA-USP7 in HEK293T cells cotransfected with HA-USP7 and Myc-DDX4. ( D ) Immunoprecipitation analysis of the interaction between endogenous USP7 and Myc-DDX4 in HEK293T cells transfected with Myc-DDX4 using either the isotype IgG (-) or anti-USP7 antibodies (USP7) for immunoprecipitation. ( E ) Immunoprecipitation analysis of the interaction between endogenous USP7 and DDX4 in HEK293T cells using either the isotype IgG (-) or anti-USP7 antibodies (USP7) for immunoprecipitation. ( F ) Western blot analysis of USP7 protein levels in HEK293T cells transfected with an increasing amount of Myc-DDX4. ( G ) Western blot analysis of HA-USP7 levels in HEK293T cells cotransfected with HA-USP7 and an increasing amount of Myc-DDX4. ( H ) Immunoprecipitation analysis of the interaction between endogenous SOCS1, endogenous IFNAR1, and HA-USP7 in HEK293T cells cotransfected with HA-USP7 and Myc-DDX4. ( I ) Immunoprecipitation analysis of the interaction between Myc-SOCS1 and HA-USP7 in HEK293T cells cotransfected with HA-USP7, Myc-SOCS1, and Myc-DDX4. ( J ) Immunoprecipitation analysis of the interaction between endogenous SOCS1 and USP7 in HEK293T cells transfected with or without Myc-DDX4. ( G ) In vitro binding assay of the interaction between SOCS1, USP7, and DDX4 by immunoprecipitating SOCS1. Data are shown as mean ± SD of three biological replicates ( B–K ).

Article Snippet: Recombinant human SOCS1 Fusion protein was purchased from Proteintech (Cat #Ag19318).

Techniques: Immunoprecipitation, Transfection, Western Blot, In Vitro, Binding Assay

Journal: mBio

Article Title: DDX4 enhances antiviral activity of type I interferon by disrupting interaction of USP7/SOCS1 and promoting degradation of SOCS1

doi: 10.1128/mbio.03213-23

Figure Lengend Snippet: DDX4 regulates SOCS1 ubiquitination levels in a USP7-dependent manner. ( A ) Immunoprecipitation analysis of polyubiquitination of Myc-SOCS1 in Ddx4 +/+ or Ddx4 -/- RAW264.7 cells cotransfected with Myc-SOCS1 and HA-Ub as indicated and then treated with MG132 (10 µM, 4 h). ( B ) Immunoprecipitation analysis of polyubiquitination of Myc-SOCS1 in Ddx4 +/+ or Ddx4 -/- RAW264.7 cells transfected with Myc-SOCS1 as indicated and then treated with MG132 (10 µM, 4 h). ( C ) Immunoprecipitation analysis of polyubiquitination of GFP-SOCS1 in HEK293T cells cotransfected with GFP-SOCS1, Myc-DDX4, and HA-Ub as indicated and then treated with MG132 (10 µM, 4 h). ( D ) Immunoprecipitation analysis of polyubiquitination of GFP-SOCS1 in HEK293T cells cotransfected with GFP-SOCS1 and Myc-DDX4 as indicated and then treated with MG132 (10 µM, 4 h). ( E ) Immunoprecipitation analysis of polyubiquitination of GFP-SOCS1 in HEK293T cells cotransfected with GFP-SOCS1, HA-Ub-K48 (R48K), and Myc-DDX4 as indicated and then treated with MG132 (10 µM, 4 h). ( F ) Immunoprecipitation analysis of polyubiquitination of GFP-SOCS1 in HEK293T cells cotransfected with GFP-SOCS1, HA-Ub-K63 (R63K), and Myc-DDX4 as indicated and then treated with MG132 (10 µM, 4 h). ( G ) Immunoprecipitation analysis of polyubiquitination of GFP-SOCS1-WT and GFP-SOCS1-K119R in HEK293T cells cotransfected with Myc-DDX4, GFP-SOCS1-WT, or GFP-SOCS1-K119R as indicated and then treated with MG132 (10 µM, 4 h). ( H ) Immunoprecipitation analysis of polyubiquitination of GFP-SOCS1 in HEK293T cells cotransfected with GFP-SOCS1, Myc-DDX4, and shUSP7 as indicated and then treated with MG132 (10 µM, 4 h). Data are shown as mean ± SD of three biological replicates ( A–G ).

Article Snippet: Recombinant human SOCS1 Fusion protein was purchased from Proteintech (Cat #Ag19318).

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Transfection

Journal: mBio

Article Title: DDX4 enhances antiviral activity of type I interferon by disrupting interaction of USP7/SOCS1 and promoting degradation of SOCS1

doi: 10.1128/mbio.03213-23

Figure Lengend Snippet: DDX4 negatively regulated the protein levels and stability of SOCS1 in a USP7-dependent manner. ( A ) Western blot analysis of SOCS1 levels in HEK293T cells transfected with an increasing amount of Myc-DDX4. ( B ) Western blot analysis of GFP-SOCS1 levels in HEK293T cells cotransfected with GFP-SOCS1 and an increasing amount of Myc-DDX4. ( C ) Western blot analysis of GFP-SOCS1 protein levels in HEK293T cells transfected with GFP-SOCS1, together with or without Myc-DDX4, followed by treatment with DMSO or MG132 (10 µM) for 12 h. ( D ) Western blot analysis of SOCS1 protein levels in RAW264.7 cells transfected with or without Myc-DDX4, followed by the treatment with CHX (50 mg/mL) as indicated. ( E ) Western blot analysis of GFP-SOCS1 protein levels in Ddx4 +/+ or Ddx4 -/- RAW264.7 cells transfected with GFP-SOCS1, followed by the treatment with CHX (50 mg/mL) as indicated. ( F ) Western blot analysis of GFP-SOCS1 protein levels in HEK293T cells cotransfected with GFP-SOCS1, shCON, shUSP7, and Myc-DDX4 as indicated. Data are shown as mean ± SD of three biological replicates ( A–F ).

Article Snippet: Recombinant human SOCS1 Fusion protein was purchased from Proteintech (Cat #Ag19318).

Techniques: Western Blot, Transfection

Journal: mBio

Article Title: DDX4 enhances antiviral activity of type I interferon by disrupting interaction of USP7/SOCS1 and promoting degradation of SOCS1

doi: 10.1128/mbio.03213-23

Figure Lengend Snippet: DDX4 regulated the antiviral function of IFN in a SOCS1/USP7-dependent manner. ( A ) Western blot analysis of PKR protein levels in HEK293T cells transfected with shCON or shUSP7, together with or without Myc-DDX4, followed by the treatment with IFNα (1,000 IU/mL) for 24 h. ( B ) Western blot analysis of PKR protein levels in HEK293T cells transfected with shCON or shSOCS1, together with or without Myc-DDX4, followed by the treatment with IFNα (1,000 IU/mL) for 24 h. ( C ) Fluorescence microscopy of VSV-GFP viruses and VSV-GFP fluorescence intensity in HEK293T cells transfected with shCON or shUSP7, together with or without Myc-DDX4. Scale bars, 100 µm. ( D ) Fluorescence microscopy of VSV-GFP viruses and VSV-GFP fluorescence intensity in HEK293T cells transfected with shCON or shSOCS1, together with or without Myc-DDX4. Scale bars, 100 µm. NS, not significant; *** P < 0.001. Data are shown as mean ± SD of three biological replicates ( A and B ).

Article Snippet: Recombinant human SOCS1 Fusion protein was purchased from Proteintech (Cat #Ag19318).

Techniques: Western Blot, Transfection, Fluorescence, Microscopy

Journal: mBio

Article Title: DDX4 enhances antiviral activity of type I interferon by disrupting interaction of USP7/SOCS1 and promoting degradation of SOCS1

doi: 10.1128/mbio.03213-23

Figure Lengend Snippet: A proposed model of regulation of the IFN-JAK/STAT1 signaling by DDX4. ( A ) USP7 physically interacts with SOCS1 and enhances SOCS1 protein stability by deubiquitination effects, which in turn restricts the IFN-I-induced activation of JAK–signal transducer. DDX4 promotes host antiviral capacity by inhibiting the interaction between USP7 and SOCS1, which subsequently promotes the degradation of SOCS1.

Article Snippet: Recombinant human SOCS1 Fusion protein was purchased from Proteintech (Cat #Ag19318).

Techniques: Activation 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: 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, Control, 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, Comparison, 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, Knockdown, 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, Knockdown, 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: Gene Expression, 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: Figure 3 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 mm. (c) Representative image of immunohistochemistry with antibodies to SOCS1 (green) and HAGE (red) on normal skin and malignant melanoma sections. Scale bar 100 mm. (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 mg, lane 2: 0.6 mg, lane 3: 1.2 mg). 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: For SOCS1 protein labelling, FM82 control cell line was transfected with SOCS1 cDNA (OriGene, SC111081) and with or without HAGE cDNA .

Techniques: Expressing, Control, Immunohistochemistry, Transfection, Knockdown, 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: Figure 4 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: For SOCS1 protein labelling, FM82 control cell line was transfected with SOCS1 cDNA (OriGene, SC111081) and with or without HAGE cDNA .

Techniques: Ubiquitin Proteomics, Control, 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: Figure 5 HAGE counteracts the anti-proliferative effect of IFNa in ABCB5 þ 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 ¼ o0.0001. Scale bar 100 mm (for spheres) and 20 mm (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 ¼ o0.0001

Article Snippet: For SOCS1 protein labelling, FM82 control cell line was transfected with SOCS1 cDNA (OriGene, SC111081) and with or without HAGE cDNA .

Techniques: Control, 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: Figure 6 HAGE counteracts the anti-proliferative effect of IFNa 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 IFNa. Student t-test: **P ¼ o0.01. (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 IFNa ANOVA: **P ¼ o0.0028; Mann–Whitney U-test: *P ¼ o0.0108 (when comparing FM82 control þ PBS and FM82 shRNA þ PBS; Mann–Whitney U-test: **P ¼ o0.0079 (when comparing FM82 control þ IFNa and FM82 shRNA þ IFNa 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 IFNa. Scale bar 100 mm

Article Snippet: For SOCS1 protein labelling, FM82 control cell line was transfected with SOCS1 cDNA (OriGene, SC111081) and with or without HAGE cDNA .

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

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: Figure 7 IFNa treatment of HAGE knocked down NOD/Scid xenotransplanted tumours results in a decreased cellularity and increased apoptosis. (a) Hematoxylin and eosin stain on sections from FM82 control and FM82 shRNA1 NOD/SCID xenotransplanted tumours and treated or untreated with IFNa. ANOVA: ***P ¼ o0.0001 (b) Immunohistochemistry/IF staining with antibody against the apoptotic marker cleaved caspase 3 on sections from FM82 control and FM82 shRNA1 NOD/SCID xenotransplanted tumours and treated or untreated with IFNa. ANOVA: ***P ¼ o0.0001. (c) Proposed schematic representation of the mechanism by which the helicase HAGE inactivate the anti-proliferative effect of IFNa in ABCB5 þ MMICs

Article Snippet: For SOCS1 protein labelling, FM82 control cell line was transfected with SOCS1 cDNA (OriGene, SC111081) and with or without HAGE cDNA .

Techniques: H&E Stain, Control, Immunohistochemistry, Staining, Marker