il rhil 22 r d systems  (R&D Systems)

Journal: Cell Communication and Signaling : CCS

Article Title: TRIM30 modulates Interleukin-22-regulated papillary thyroid Cancer cell migration and invasion by targeting Sox17 for K48-linked Polyubiquitination

doi: 10.1186/s12964-019-0484-6

Figure Lengend Snippet: TRIM30 associates with Sox17. ( a ) TPC-1 cells were transfected with vector control or Myc-Sox17 for 48 h. Cells were lysed and immunopurified with anti-Flag affinity columns and eluted with Flag peptide. The eluates were resolved using SDS-PAGE and were silver-stained. The various protein bands were retrieved and analyzed using mass spectrometry. ( b ) 293 T cells were transfected with Flag-tagged TRIM30 (Flag-TRIM30) and Myc-tagged Sox17 (Myc- Sox17). Forty-eight hours post-transfection, Co-IP and immunoblot analysis were performed with the indicated antibodies. ( c ) Experiments were performed in similar fashion to those in ( b ), except DNAase was used. ( d ) Experiments were performed in similar fashion to those in ( b ), except Flag-TRIM8 was used. ( e ) Schematic diagram of the full-length and truncated constructs of Sox17 (upper panel). 293 T cells were co-transfected with Flag-TRIM30 and the indicated truncated Sox17 constructs for 48 h. Co-IP and immunoblot analyses were performed with the indicated antibodies (lower panel). ( f ) Experiments were performed in similar fashion to those in ( e ), except indicated truncated constructs of TRIM30 were used. ( g ) TPC-1 cells were treated with rhIL-22 at 50 ng/ml for indicated times. Immunoprecipitation and immunoblot analysis were performed with the indicated antibodies. All experiments were repeated at least three times

Article Snippet: Recombinant human IL-22 (rhIL-22) was purchased from R&D Systems (Minneapolis, MN).

Techniques: Transfection, Plasmid Preparation, SDS Page, Staining, Mass Spectrometry, Co-Immunoprecipitation Assay, Western Blot, Construct, Immunoprecipitation

Journal: Cell Communication and Signaling : CCS

Article Title: TRIM30 modulates Interleukin-22-regulated papillary thyroid Cancer cell migration and invasion by targeting Sox17 for K48-linked Polyubiquitination

doi: 10.1186/s12964-019-0484-6

Figure Lengend Snippet: TRIM30 is involved in IL-22 regulated K48-linked polyubiquitination of Sox17. ( a ) TPC-1 cells were treated with 50 ng/ml rhIL-22 for the indicated time points. TRIM30 RNA levels were quantified using qRT-PCR (upper panel) and protein levels of TRIM30 and Sox17 were measured using western blot (lower panel). ( b ) TPC-1 cells were treated with rhIL-22 for 24 h at indicated concentrations. TRIM30 RNA levels were quantified using qRT-PCR (upper panel) and protein levels of TRIM30 and Sox17 were measured using western blot (lower panel). ( c ) TPC-1 cells were transfected with indicated plasmids for 24 h and treated with or without 50 ng/ml rhIL- 22 for 24 h prior to real-time RT-PCR (upper panel) and western blot (lower panel). ( d ) Experiments were performed as in ( c ) except cells were transfected with TRIM30-specific shRNA (shRNA-TRIM30). ( e ) TPC-1 cells were transfected with pCMV-TRIM30 or control vector for 48 h and treated with or without cycloheximide (CHX) for indicated times prior to western blot. ( f ) Overexpressed TRIM30 enhances wild-type and K48-linked polyubiquitination of Sox17. A total of 293 cells (2 × 10 6 ) were transfected with Sox17 or/and Flag-TRIM30 and the indicated ubiquitin plasmids for 24 h and treated with MG132 (100 μM) for 6 h. Cell lysates were immunoprecipitated with anti-Myc. The immunoprecipitates were analyzed by immunoblots with anti-HA, anti-Myc, or anti-Flag as indicated. ( g ) TPC-1 cells were treated with 50 ng/ml rhIL-22 for the indicated time points. Co-IP and immunoblot analyses were performed with the indicated antibodies. ( h ) TPC-1 cells were transfected with indicated plasmid for 24 h and treated with or without 50 ng/ml rhIL- 22 for 24 h. Co-IP and immunoblot analyses were performed with the indicated antibodies. ( i ) Experiments were performed as in ( c ) except cells were transfected with shRNA-TRIM30. All experiments were repeated at least three times. Bar graphs present means ± SD, n = 3 (** P < 0.01; * P < 0.05)

Article Snippet: Recombinant human IL-22 (rhIL-22) was purchased from R&D Systems (Minneapolis, MN).

Techniques: Quantitative RT-PCR, Western Blot, Transfection, shRNA, Plasmid Preparation, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Cell Communication and Signaling : CCS

Article Title: TRIM30 modulates Interleukin-22-regulated papillary thyroid Cancer cell migration and invasion by targeting Sox17 for K48-linked Polyubiquitination

doi: 10.1186/s12964-019-0484-6

Figure Lengend Snippet: IL-22 promotes cell growth and motility via TRIM30/Sox17 axis. (A) TPC-1 cells transfected with the pCMV-TRIM30 or control vector for 24 h and treated with or without 50 ng/ml rhIL- 22 for 24 h prior to cell proliferation assay. (B) Experiments were performed as in ( a ), except cells were transfected with shRNA- TRIM30. (C and D) TPC-1 cells were transfected with the indicated plasmid, then treated with rhIL-22 (50 ng/ml) for 48 h prior to Transwell migration ( c ) or invasion ( d ) assays. ( e and f ) Experiments were performed as in ( c and d ), except cells were transfected with shRNA-TRIM30. (G) TPC-1 cells were transfected with the plasmids as indicated for 48 h prior to cell proliferation assay. ( i ) Experiments were performed as in ( g ), except cells were transfected with shRNA-Sox17. (I and J) TPC-1 cells were transfected with the plasmids as indicated for 48 h prior to Transwell migration ( i ) or invasion ( j ) assays. ( k and l ) Experiments were performed as in (I and J), except cells were transfected with shRNA-Sox17. (M-P) TPC-1 cells were stably expressing TRIM30, SOX17, or TRIM30 and SOX17 were subcutaneous injected into nude mice. Shown are stripped tumors ( m ), the growth curve ( n ), and the average weight ( o ). The indicated tumor tissue from a single mouse in each group was cut into several pieces and then used for immunoblot ( p ). Tumors were from the fourth column in each group. Bar graphs present means ± SD, n = 3 (** P < 0.01; * P < 0.05)

Article Snippet: Recombinant human IL-22 (rhIL-22) was purchased from R&D Systems (Minneapolis, MN).

Techniques: Transfection, Plasmid Preparation, Proliferation Assay, shRNA, Migration, Stable Transfection, Expressing, Injection, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: TRIM30 modulates Interleukin-22-regulated papillary thyroid Cancer cell migration and invasion by targeting Sox17 for K48-linked Polyubiquitination

doi: 10.1186/s12964-019-0484-6

Figure Lengend Snippet: IL-22 regulate β-catenin signaling via TRIM30/Sox17 axis. ( a ) TPC-1 cells were treated with a concentration of 50 ng/ml rhIL-22 for the indicated time points. β-catenin levels were quantified by qRT-PCR (upper panel) and western blot (lower panel). ( b ) TPC-1 cells were treated with rhIL-22 for 24 h at indicated concentrations. β-catenin levels were quantified by qRT-PCR (upper panel) and western blot (lower panel). ( c ) TPC-1 cells transfected with the indicated plasmid for 24 h and treated with or without 50 ng/ml rhIL- 22 for 24 h. Co-IP and immunoblot analyses were performed with the indicated antibodies. ( d ) TPC-1 cells transfected with the indicated plasmid for 24 h and treated with or without 50 ng/ml rhIL- 22 for 24 h prior to luciferase assays. ( e ) Experiments were performed as in ( d ), except cells were transfected with shRNA-Sox17. (f) TPC-1 cells transfected with the indicated plasmid for 24 h and treated with or without 50 ng/ml rhIL- 22 for 24 h. Cytosolic and nuclear extracts were prepared and subjected to western blot analyses. Lamin A and β-actin were used as markers for nuclear and cytosolic fractions, respectively. ( g ) Experiments were performed as in ( f ), except cells were transfected with shRNA-Sox17. ( h ) Experiments were performed as in ( c ), except TRIM −/− cells were used. ( i and j ) Experiments were performed as in ( d and e ), except pCMV-TRIM30 (i) or shRNA-TRIM30 ( j ) were used. ( k and l ) Experiments were performed as in (f and g), except pCMV-TRIM30 ( k ) or shRNA-TRIM30 (l) were used. All experiments were repeated at least three times. Bar graphs present means ± SD, n = 3 (** P < 0.01; * P < 0.05)

Article Snippet: Recombinant human IL-22 (rhIL-22) was purchased from R&D Systems (Minneapolis, MN).

Techniques: Concentration Assay, Quantitative RT-PCR, Western Blot, Transfection, Plasmid Preparation, Co-Immunoprecipitation Assay, Luciferase, shRNA

Journal: Cell Communication and Signaling : CCS

Article Title: TRIM30 modulates Interleukin-22-regulated papillary thyroid Cancer cell migration and invasion by targeting Sox17 for K48-linked Polyubiquitination

doi: 10.1186/s12964-019-0484-6

Figure Lengend Snippet: Expression IL-22, TRIM30, and β-catenin in papillary thyroid cancer tissues. ( a-c ) qRT-PCR experiments analyzing the expression of IL-22 ( a ), TRIM30 ( b ), and β-catenin ( c ) in 116 PTCs in comparison with the mean value obtained from 116 normal thyroid samples. ( d-f ) The relative IL-22 mRNA and TRIM30 levels ( d ), the relative IL-22 mRNA and β-catenin levels ( e ) and the relative TRIM30 mRNA and β-catenin levels ( f ) in the PTCs were subjected to Pearson’s correlation analysis. Box plots illustrate medians with 25th and 75th percentiles and error bars for 5th and 95th percentiles. For A-C, the lowest value was designated as 1. IL-22, TRIM30, and β-catenin data are expressed as fold-induction relative to the lowest value (** P < 0.01; * P < 0.05). The p values were calculated in SPSS 17.0 using Student’s t test

Article Snippet: Recombinant human IL-22 (rhIL-22) was purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Quantitative RT-PCR, Comparison

Journal: Cell Communication and Signaling : CCS

Article Title: TRIM30 modulates Interleukin-22-regulated papillary thyroid Cancer cell migration and invasion by targeting Sox17 for K48-linked Polyubiquitination

doi: 10.1186/s12964-019-0484-6

Figure Lengend Snippet: A hypothetical model for the role of TRIM30 in IL-22-regulated PTC cell migration and invasion. Under IL-22 treatment, TRIM30 associates with Sox17 for K48-linked polyubiquitination, thereby impairing the interaction between Sox17 and β-catenin. As a result, β-catenin translocates from the cytosol to the nucleus and activates downstream signaling, consequently contributing to PTC development

Article Snippet: Recombinant human IL-22 (rhIL-22) was purchased from R&D Systems (Minneapolis, MN).

Techniques: Migration

Journal: Molecular Medicine

Article Title: Interleukin-26 Production in Human Primary Bronchial Epithelial Cells in Response to Viral Stimulation: Modulation by Th17 cytokines

doi: 10.2119/molmed.2016.00064

Figure Lengend Snippet: Th17 cytokines modulate poly-IC induced release of IL-26. Primary bronchial epithelial cells were stimulated with poly-IC (0.05 μg/mL) in the presence or absence of rhIL-17A (100 ng/mL) or rhIL-22 (100 ng/mL) or rhIL-17A (100 ng/mL) plus rhIL-22 (100 ng/mL) (24 h). Extracellular concentrations of IL-26 in cell-free conditioned media as well as intracellular levels were measured using ELISA and western blot respectively and mRNA level by real time PCR. (A) Extracellular concentrations of IL-26 in cell-free conditioned media in response to rhIL-17A and/or rhIL-22 (n = 14). (B) IL26 mRNA level in response to rhIL-17A (n = 9). (C) Extracellular concentrations of IL-26 in cell-free conditioned media in response to poly-IC plus rhIL-17A (n = 6). (D) Extracellular concentrations of IL-26 in cell-free conditioned media in response to poly-IC plus IL-22 (n = 6). (E) Extracellular concentrations of IL-26 in cell-free conditioned media in response to poly-IC plus rhIL-17A plus rhIL-22. (n = 6). (F) Intracellular IL-26 protein (representative western blot) and (G) the average protein level (fold difference) in response to IL-17A during 24 h (n = 10). The p values indicated are cording to the Student paired t test. p < 0.05 is considered significant

Article Snippet: Semiconfluent cells were stimulated (1 mL of culture media) with different concentrations of the TLR3 agonist poly-IC, as well as additional types of viral stimulation including the TLR7 agonist Imiquimod, and the TLR8 agonist single stranded (ss) RNA (Invivogen ® ) ( 14 , 16 ) as well as recombinant human (rh) Th17 cytokine proteins including rhIL-17A and rhIL-22 (R&D systems).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Real-time Polymerase Chain Reaction