Journal: Arthritis

Article Title: Combination of Interleukin-27 and MicroRNA for Enhancing Expression of Anti-Inflammatory and Proosteogenic Genes

doi: 10.1155/2017/6365857

Figure Lengend Snippet: IL-27 effect on osteoblast and osteoclast cell signaling. (a) IL-27 upregulates expression of several genes related to osteoblast differentiation pathways. Expression changes were determined by utilizing a luciferase reporter assay for indicating activity of BMP, WNT, STAT1, STAT3, OPN, RUNX2, and OSX promoters. Black rectangle frames (□) highlight the data with significant expression changes relative to untreated or negative control (pOB−ctrl); +OB ctrl, differentiating MC3T3E1-14 osteoblasts treated with differentiation supplements for 72 h. Right plot , IL-27 upregulates osteoblast differentiation genes and downregulates osteoblast inhibitor genes, as assessed by quantitative real time (qPCR) assays. ( ∗ P < 0.05 relative to untreated or negative control, pOB−ctrl). (b) IL-27 downregulates proinflammatory genes in fibroblasts and differentiation genes in osteoclasts, as assessed by quantitative real time (qPCR) assays ( ∗ P < 0.05 versus untreated controls). Right plot, IL-27 treatment downregulates several pathways associated with osteoclast activity as assessed by luciferase reporter assay for indicating activity of TNF, IL-17a, TRAF2, STAT3, and STAT1 promoters. Black rectangle frames (□) highlight the data with significant expression changes ( P < 0.05) relative to untreated or negative control (pOB−ctrl); +OB ctrl, differentiating MC3T3E1-14 osteoblasts treated with differentiation supplements for the days indicated.

Article Snippet: For luciferase assays, constructs responsive to the active (phosphorylated) form of STAT1 or STAT3 were used (STAT1.GAS/ISRE- and STAT3/3-Luc; Panomics, Fremont, CA) or OPN-, RUNX2-, IL-17a-, TRAF2-, and TLR10-Luc (Switchgear Genomics, Carlsbad, CA) and Tcf/Lef-, BRE-, and SBE/FBE-Luc (SBE4-) or TNF-Luc (12456, 45126, 16525, and 11110, Addgene, Cambridge, MA) and 2% CMV-Bgal (Clontech, Mountain View, CA) as a transfection control to transfect cells using Lipofectamine 3000 or DMRIE-C reagents (Invitrogen, Carlsbad, CA) according to manufacturer's protocols [ ]. miRNA mimics were purchased from Sigma-Aldrich at 5 nmol and utilized in a transfection at a 5 pmol final concentration. miRNA sequences are 100% conserved in human and mouse miRNA according to the miRbase database [ ] and are as follows, miR-17, MIMAT0000070, CAAAGUGCUUACAGUGCAGGUAG , miR-210, MIMAT0000267, CUGUGCGUGUGACAGCGGCUGA , miR-29b-3p, MIMAT0000100, UAGCACCAUUUGAAAUCAGUGUU, miR-20b, MIMAT0001413, CAAAGUGCUCAUAGUGCAGGUAG , miR-10a, MIMAT0000253, UACCCUGUAGAUCCGAAUUUGUG , miR-let7f-5p, MIMAT0000067, UGAGGUAGUAGAUUGUAUAGUU, and miR-21-5p, MIMAT0000076, UAGCUUAUCAGACUGAUGUUGA .

Techniques: Expressing, Luciferase, Reporter Assay, Activity Assay, Negative Control

Journal: Arthritis

Article Title: Combination of Interleukin-27 and MicroRNA for Enhancing Expression of Anti-Inflammatory and Proosteogenic Genes

doi: 10.1155/2017/6365857

Figure Lengend Snippet: Osteoblastic cells. MC3T3E1(14) cells cultured in the presence of ascorbic acid and beta-glycerol phosphate were cotransfected with miR control or experimental (miRlet7f, miR-10a, miR-20b, miR-21, miR-210, miR-17, and miR-29b) and luciferase reporter vectors for detecting IL17a, TNF, and TRAF2 expression or SMAD2/3, STAT3, and STAT1 activity. (a) Effect of IL-27 (50 ng/ml) on luciferase expression. (b) Effect of experimental miRNA on luciferase expression. (c) Effect of miRNA+IL27 combination on luciferase expression. Black rectangle frames (□) highlight the data with significant expression changes ( P < 0.05) relative to negative control (miR ctrl); osteoblastic cells, differentiating MC3T3E1-14 cells treated with differentiation supplements for 7 days. Yellow arrowheads, proposed best combinations of miR and IL-27. Color bar, range of fold change in expression, from green (−4.0-fold change or decrease) to red (2.8-fold change or increase).

Article Snippet: For luciferase assays, constructs responsive to the active (phosphorylated) form of STAT1 or STAT3 were used (STAT1.GAS/ISRE- and STAT3/3-Luc; Panomics, Fremont, CA) or OPN-, RUNX2-, IL-17a-, TRAF2-, and TLR10-Luc (Switchgear Genomics, Carlsbad, CA) and Tcf/Lef-, BRE-, and SBE/FBE-Luc (SBE4-) or TNF-Luc (12456, 45126, 16525, and 11110, Addgene, Cambridge, MA) and 2% CMV-Bgal (Clontech, Mountain View, CA) as a transfection control to transfect cells using Lipofectamine 3000 or DMRIE-C reagents (Invitrogen, Carlsbad, CA) according to manufacturer's protocols [ ]. miRNA mimics were purchased from Sigma-Aldrich at 5 nmol and utilized in a transfection at a 5 pmol final concentration. miRNA sequences are 100% conserved in human and mouse miRNA according to the miRbase database [ ] and are as follows, miR-17, MIMAT0000070, CAAAGUGCUUACAGUGCAGGUAG , miR-210, MIMAT0000267, CUGUGCGUGUGACAGCGGCUGA , miR-29b-3p, MIMAT0000100, UAGCACCAUUUGAAAUCAGUGUU, miR-20b, MIMAT0001413, CAAAGUGCUCAUAGUGCAGGUAG , miR-10a, MIMAT0000253, UACCCUGUAGAUCCGAAUUUGUG , miR-let7f-5p, MIMAT0000067, UGAGGUAGUAGAUUGUAUAGUU, and miR-21-5p, MIMAT0000076, UAGCUUAUCAGACUGAUGUUGA .

Techniques: Cell Culture, Luciferase, Expressing, Activity Assay, Negative Control

Journal: Arthritis

Article Title: Combination of Interleukin-27 and MicroRNA for Enhancing Expression of Anti-Inflammatory and Proosteogenic Genes

doi: 10.1155/2017/6365857

Figure Lengend Snippet: Osteoclastic cells. RAW264.7 cells cultured in the presence of 35 ng/ml RANKL and cotransfected with miR control or experimental (miRlet7f, miR-10a, miR-20b, miR-21, miR-29b, miR-17, and miR-210) and luciferase reporter vectors for detecting IL17a, TNF, and TRAF2 expression or SMAD2/3, STAT3, and STAT1 activity. (a) Effect of IL-27 (50 ng/ml) on luciferase expression. (b) Effect of experimental miRNA on luciferase expression. (c) Effect of miRNA+IL27 combination on luciferase expression. Black rectangle frames (□) highlight the data with significant expression changes ( P < 0.05) relative to negative control (miR ctrl); osteoclasts, differentiating RAW264.7 cells treated with differentiation supplements for 6 days. Yellow arrowheads, proposed best combinations of miR and IL-27. Color bar, range of fold change in expression, from green (−2.0-fold change or decrease) to red (2.1-fold change or increase).

Article Snippet: For luciferase assays, constructs responsive to the active (phosphorylated) form of STAT1 or STAT3 were used (STAT1.GAS/ISRE- and STAT3/3-Luc; Panomics, Fremont, CA) or OPN-, RUNX2-, IL-17a-, TRAF2-, and TLR10-Luc (Switchgear Genomics, Carlsbad, CA) and Tcf/Lef-, BRE-, and SBE/FBE-Luc (SBE4-) or TNF-Luc (12456, 45126, 16525, and 11110, Addgene, Cambridge, MA) and 2% CMV-Bgal (Clontech, Mountain View, CA) as a transfection control to transfect cells using Lipofectamine 3000 or DMRIE-C reagents (Invitrogen, Carlsbad, CA) according to manufacturer's protocols [ ]. miRNA mimics were purchased from Sigma-Aldrich at 5 nmol and utilized in a transfection at a 5 pmol final concentration. miRNA sequences are 100% conserved in human and mouse miRNA according to the miRbase database [ ] and are as follows, miR-17, MIMAT0000070, CAAAGUGCUUACAGUGCAGGUAG , miR-210, MIMAT0000267, CUGUGCGUGUGACAGCGGCUGA , miR-29b-3p, MIMAT0000100, UAGCACCAUUUGAAAUCAGUGUU, miR-20b, MIMAT0001413, CAAAGUGCUCAUAGUGCAGGUAG , miR-10a, MIMAT0000253, UACCCUGUAGAUCCGAAUUUGUG , miR-let7f-5p, MIMAT0000067, UGAGGUAGUAGAUUGUAUAGUU, and miR-21-5p, MIMAT0000076, UAGCUUAUCAGACUGAUGUUGA .

Techniques: Cell Culture, Luciferase, Expressing, Activity Assay, Negative Control

Journal: Arthritis

Article Title: Combination of Interleukin-27 and MicroRNA for Enhancing Expression of Anti-Inflammatory and Proosteogenic Genes

doi: 10.1155/2017/6365857

Figure Lengend Snippet: Macrophage cells. RAW264.7 macrophages cultured in the presence of proinflammatory LPS stimulus and cotransfected with miR control or experimental (miRlet7f, miR-10a, miR-20b, miR-21, miR-29b, miR-17, and miR-210) and luciferase reporter vectors for detecting IL17a, TNF, and TRAF2 expression or SMAD2/3, STAT3, and STAT1 activity. (a) Effect of IL-27 (50 ng/ml) on luciferase expression. (b) Effect of experimental miRNA on luciferase expression. (c) Effect of miRNA+IL27 combination on luciferase expression. Black rectangle frames (□) highlight the data with significant expression changes ( P < 0.05) relative to negative control (miR ctrl); macrophages, RAW264.7 cells treated with activating agent LPS as per Materials and Methods. Yellow arrowheads, proposed best combinations of miR and IL-27. Color bar, range of fold change in expression, from green (−2.0-fold change or decrease) to red (2.4-fold change or increase).

Article Snippet: For luciferase assays, constructs responsive to the active (phosphorylated) form of STAT1 or STAT3 were used (STAT1.GAS/ISRE- and STAT3/3-Luc; Panomics, Fremont, CA) or OPN-, RUNX2-, IL-17a-, TRAF2-, and TLR10-Luc (Switchgear Genomics, Carlsbad, CA) and Tcf/Lef-, BRE-, and SBE/FBE-Luc (SBE4-) or TNF-Luc (12456, 45126, 16525, and 11110, Addgene, Cambridge, MA) and 2% CMV-Bgal (Clontech, Mountain View, CA) as a transfection control to transfect cells using Lipofectamine 3000 or DMRIE-C reagents (Invitrogen, Carlsbad, CA) according to manufacturer's protocols [ ]. miRNA mimics were purchased from Sigma-Aldrich at 5 nmol and utilized in a transfection at a 5 pmol final concentration. miRNA sequences are 100% conserved in human and mouse miRNA according to the miRbase database [ ] and are as follows, miR-17, MIMAT0000070, CAAAGUGCUUACAGUGCAGGUAG , miR-210, MIMAT0000267, CUGUGCGUGUGACAGCGGCUGA , miR-29b-3p, MIMAT0000100, UAGCACCAUUUGAAAUCAGUGUU, miR-20b, MIMAT0001413, CAAAGUGCUCAUAGUGCAGGUAG , miR-10a, MIMAT0000253, UACCCUGUAGAUCCGAAUUUGUG , miR-let7f-5p, MIMAT0000067, UGAGGUAGUAGAUUGUAUAGUU, and miR-21-5p, MIMAT0000076, UAGCUUAUCAGACUGAUGUUGA .

Techniques: Cell Culture, Luciferase, Expressing, Activity Assay, Negative Control

Journal: Arthritis

Article Title: Combination of Interleukin-27 and MicroRNA for Enhancing Expression of Anti-Inflammatory and Proosteogenic Genes

doi: 10.1155/2017/6365857

Figure Lengend Snippet: Fibroblastic cells. NIH 3T3 fibroblastic cells grown in the presence of TNF stimulus and cotransfected with miR control or experimental (miRlet7f, miR-10a, miR-20b, or miR-21) and luciferase reporter vectors for detecting IL17a and TNF expression or STAT3 and STAT1 activity. (a) Effect of IL-27 (50 ng/ml) on luciferase expression. (b) Effect of experimental miRNA on luciferase expression. (c) Effect of miRNA+IL27 combination on luciferase expression. Black rectangle frames (□) highlight the data with significant expression changes ( P < 0.05) relative to negative control (miR ctrl); fibroblastic cells, NIH3T3 cells treated with activating agent TNF as per Materials and Methods. Yellow arrowheads, proposed best combinations of miR and IL-27. Color bar, range of fold change in expression, from green (−2.2-fold change or decrease) to red (2.2-fold change or increase).

Article Snippet: For luciferase assays, constructs responsive to the active (phosphorylated) form of STAT1 or STAT3 were used (STAT1.GAS/ISRE- and STAT3/3-Luc; Panomics, Fremont, CA) or OPN-, RUNX2-, IL-17a-, TRAF2-, and TLR10-Luc (Switchgear Genomics, Carlsbad, CA) and Tcf/Lef-, BRE-, and SBE/FBE-Luc (SBE4-) or TNF-Luc (12456, 45126, 16525, and 11110, Addgene, Cambridge, MA) and 2% CMV-Bgal (Clontech, Mountain View, CA) as a transfection control to transfect cells using Lipofectamine 3000 or DMRIE-C reagents (Invitrogen, Carlsbad, CA) according to manufacturer's protocols [ ]. miRNA mimics were purchased from Sigma-Aldrich at 5 nmol and utilized in a transfection at a 5 pmol final concentration. miRNA sequences are 100% conserved in human and mouse miRNA according to the miRbase database [ ] and are as follows, miR-17, MIMAT0000070, CAAAGUGCUUACAGUGCAGGUAG , miR-210, MIMAT0000267, CUGUGCGUGUGACAGCGGCUGA , miR-29b-3p, MIMAT0000100, UAGCACCAUUUGAAAUCAGUGUU, miR-20b, MIMAT0001413, CAAAGUGCUCAUAGUGCAGGUAG , miR-10a, MIMAT0000253, UACCCUGUAGAUCCGAAUUUGUG , miR-let7f-5p, MIMAT0000067, UGAGGUAGUAGAUUGUAUAGUU, and miR-21-5p, MIMAT0000076, UAGCUUAUCAGACUGAUGUUGA .

Techniques: Luciferase, Expressing, Activity Assay, Negative Control

Journal: Arthritis

Article Title: Combination of Interleukin-27 and MicroRNA for Enhancing Expression of Anti-Inflammatory and Proosteogenic Genes

doi: 10.1155/2017/6365857

Figure Lengend Snippet: Jurkat T cells. T cells were grown in the absence (a–c) or presence (d–f) of PMA/Ionomycin stimulus. Cells were cotransfected with miR control or experimental (miRlet7f, miR-10a, miR-20b, or miR-21) and luciferase reporter vectors for detecting IL17a, TNF, and TLR10 expression or STAT3 and STAT1 activity. (a) Effect of IL-27 (50 ng/ml) on luciferase expression. (b) Effect of experimental miRNA on luciferase expression. (c) Effect of miRNA+IL27 combination on luciferase expression. Black rectangle frames (□) highlight the data with significant expression changes ( P < 0.05) relative to negative control (miR ctrl); Jurkat T cells cultured in the absence (a–c) or presence of PMA/I (d–f) as per Materials and Methods. Yellow arrowheads, proposed best combinations of miR and IL-27. Color bars, range of fold change in expression, from green (−2.4- or −4.9-fold change or decrease) to red (2.5- or 3.7-fold change or increase).

Article Snippet: For luciferase assays, constructs responsive to the active (phosphorylated) form of STAT1 or STAT3 were used (STAT1.GAS/ISRE- and STAT3/3-Luc; Panomics, Fremont, CA) or OPN-, RUNX2-, IL-17a-, TRAF2-, and TLR10-Luc (Switchgear Genomics, Carlsbad, CA) and Tcf/Lef-, BRE-, and SBE/FBE-Luc (SBE4-) or TNF-Luc (12456, 45126, 16525, and 11110, Addgene, Cambridge, MA) and 2% CMV-Bgal (Clontech, Mountain View, CA) as a transfection control to transfect cells using Lipofectamine 3000 or DMRIE-C reagents (Invitrogen, Carlsbad, CA) according to manufacturer's protocols [ ]. miRNA mimics were purchased from Sigma-Aldrich at 5 nmol and utilized in a transfection at a 5 pmol final concentration. miRNA sequences are 100% conserved in human and mouse miRNA according to the miRbase database [ ] and are as follows, miR-17, MIMAT0000070, CAAAGUGCUUACAGUGCAGGUAG , miR-210, MIMAT0000267, CUGUGCGUGUGACAGCGGCUGA , miR-29b-3p, MIMAT0000100, UAGCACCAUUUGAAAUCAGUGUU, miR-20b, MIMAT0001413, CAAAGUGCUCAUAGUGCAGGUAG , miR-10a, MIMAT0000253, UACCCUGUAGAUCCGAAUUUGUG , miR-let7f-5p, MIMAT0000067, UGAGGUAGUAGAUUGUAUAGUU, and miR-21-5p, MIMAT0000076, UAGCUUAUCAGACUGAUGUUGA .

Techniques: Luciferase, Expressing, Activity Assay, Negative Control, Cell Culture

Journal: Arthritis

Article Title: Combination of Interleukin-27 and MicroRNA for Enhancing Expression of Anti-Inflammatory and Proosteogenic Genes

doi: 10.1155/2017/6365857

Figure Lengend Snippet: EL4 T cells. T cells were grown in the absence (a–c) or presence (d–f) of PMA/Ionomycin stimulus. Cells were cotransfected with miR control or experimental (miRlet7f, miR-10a, miR-20b, or miR-21) and luciferase reporter vectors for detecting IL17a, TNF, and TLR10 expression or STAT3 and STAT1 activity. (a) Effect of IL-27 (50 ng/ml) on luciferase expression. (b) Effect of experimental miRNA on luciferase expression. (c) Effect of miRNA+IL27 combination on luciferase expression. Black rectangle frames (□) highlight the data with significant expression changes ( P < 0.05) relative to negative control (miR ctrl); EL4 T cells cultured in the absence (a–c) or presence of PMA/I (d–f) as per Materials and Methods. Yellow arrowheads, proposed best combinations of miR and IL-27. Color bar, range of fold change in expression, from green (−2.4- or −2.6-fold change or decrease) to red (12.0- or 16.7-fold change or increase).

Article Snippet: For luciferase assays, constructs responsive to the active (phosphorylated) form of STAT1 or STAT3 were used (STAT1.GAS/ISRE- and STAT3/3-Luc; Panomics, Fremont, CA) or OPN-, RUNX2-, IL-17a-, TRAF2-, and TLR10-Luc (Switchgear Genomics, Carlsbad, CA) and Tcf/Lef-, BRE-, and SBE/FBE-Luc (SBE4-) or TNF-Luc (12456, 45126, 16525, and 11110, Addgene, Cambridge, MA) and 2% CMV-Bgal (Clontech, Mountain View, CA) as a transfection control to transfect cells using Lipofectamine 3000 or DMRIE-C reagents (Invitrogen, Carlsbad, CA) according to manufacturer's protocols [ ]. miRNA mimics were purchased from Sigma-Aldrich at 5 nmol and utilized in a transfection at a 5 pmol final concentration. miRNA sequences are 100% conserved in human and mouse miRNA according to the miRbase database [ ] and are as follows, miR-17, MIMAT0000070, CAAAGUGCUUACAGUGCAGGUAG , miR-210, MIMAT0000267, CUGUGCGUGUGACAGCGGCUGA , miR-29b-3p, MIMAT0000100, UAGCACCAUUUGAAAUCAGUGUU, miR-20b, MIMAT0001413, CAAAGUGCUCAUAGUGCAGGUAG , miR-10a, MIMAT0000253, UACCCUGUAGAUCCGAAUUUGUG , miR-let7f-5p, MIMAT0000067, UGAGGUAGUAGAUUGUAUAGUU, and miR-21-5p, MIMAT0000076, UAGCUUAUCAGACUGAUGUUGA .

Techniques: Luciferase, Expressing, Activity Assay, Negative Control, Cell Culture