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Fig. 7. miR-155 targets SOCS-1 to regulate inflammatory response in macrophages. A: wild-type 3=-UTR of mouse SOCS-1 (SOCS-1 3=-UTR) or miR-155 binding deficient mutant 3=-UTR of mouse SOCS-1 (mutSOCS-1 3=-UTR) was separately inserted into the pMIR-REPORT miRNA expression reporter vector. Then these vectors were combined with either miR-155 mimics mmu-miR-155-5p miRNA precursor (Pre-miR-155) or miRNA precursor negative control (Scramble) to be cotransfected into HEK-293T cells. The luciferase activity of the reporter vector was assayed, and the relative activity was compared between groups. n 3 samples per group. *P 0.05 vs. control miRNA and SOCS-1 3=-UTR cotransfected group. B: BMDMs were transfected with SOCS-1 siRNA or control nonsense siRNA. Protein levels of SOCS-1 in cell lysates after transfection were assayed by <t>Western</t> <t>blot</t> <t>analysis.</t> C and D: BMDMs were differentiated from miR-155/ mice or WT control mice, respectively. Then these cells were transfected with either SOCS-1 siRNA or control nonsense siRNA. Immediately after transfection, BMDMs were treated with LPS (100 ng/ml) for 6 h, and mRNA levels of TNF- (C) and IL-6 (D) were assayed by real-time PCR analysis. n 3 samples per group.*P 0.05 vs. BMDMs from WT mice transfected with control siRNA. #P 0.05 vs. BMDMs from WT mice transfected with SOCS-1 siRNA. E: miR-155/ BMDMs transfected with either Pre-miR-155 or miRNA Scramble Control. Then, these cells were transfected with lentivirus expressing SOCS-1 cDNA tagged with a mutated SOCS-1 3=UTR (mutSCOS-1), which was unable to bind miR-155. An empty lentivirus vector (lentiviral vector) was used as a control. At 72 h after transfection, protein levels of SOCS-1 in transfected cells were assayed by Western blot analysis. F and G: 72 h after BMDMs were transfected with indicated miRNAs and lentivirus, BMDMs were stimulated with LPS for 6 h. mRNA levels of TNF- (F), and IL-6 (G) were assayed by real-time PCR analysis. n 3 samples per group. *P 0.05 vs. BMDMs transfected with Pre-miR-155 and lentiviral vector. #P 0.05 vs. BMDMs transfected with Scramble and mutSOCS-1. §P 0.05 vs. BMDMs transfected with Scramble and lentiviral vector.
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Fig. 7. miR-155 targets SOCS-1 to regulate inflammatory response in macrophages. A: wild-type 3=-UTR of mouse SOCS-1 (SOCS-1 3=-UTR) or miR-155 binding deficient mutant 3=-UTR of mouse SOCS-1 (mutSOCS-1 3=-UTR) was separately inserted into the pMIR-REPORT miRNA expression reporter vector. Then these vectors were combined with either miR-155 mimics mmu-miR-155-5p miRNA precursor (Pre-miR-155) or miRNA precursor negative control (Scramble) to be cotransfected into HEK-293T cells. The luciferase activity of the reporter vector was assayed, and the relative activity was compared between groups. n 3 samples per group. *P 0.05 vs. control miRNA and SOCS-1 3=-UTR cotransfected group. B: BMDMs were transfected with SOCS-1 siRNA or control nonsense siRNA. Protein levels of SOCS-1 in cell lysates after transfection were assayed by <t>Western</t> <t>blot</t> <t>analysis.</t> C and D: BMDMs were differentiated from miR-155/ mice or WT control mice, respectively. Then these cells were transfected with either SOCS-1 siRNA or control nonsense siRNA. Immediately after transfection, BMDMs were treated with LPS (100 ng/ml) for 6 h, and mRNA levels of TNF- (C) and IL-6 (D) were assayed by real-time PCR analysis. n 3 samples per group.*P 0.05 vs. BMDMs from WT mice transfected with control siRNA. #P 0.05 vs. BMDMs from WT mice transfected with SOCS-1 siRNA. E: miR-155/ BMDMs transfected with either Pre-miR-155 or miRNA Scramble Control. Then, these cells were transfected with lentivirus expressing SOCS-1 cDNA tagged with a mutated SOCS-1 3=UTR (mutSCOS-1), which was unable to bind miR-155. An empty lentivirus vector (lentiviral vector) was used as a control. At 72 h after transfection, protein levels of SOCS-1 in transfected cells were assayed by Western blot analysis. F and G: 72 h after BMDMs were transfected with indicated miRNAs and lentivirus, BMDMs were stimulated with LPS for 6 h. mRNA levels of TNF- (F), and IL-6 (G) were assayed by real-time PCR analysis. n 3 samples per group. *P 0.05 vs. BMDMs transfected with Pre-miR-155 and lentiviral vector. #P 0.05 vs. BMDMs transfected with Scramble and mutSOCS-1. §P 0.05 vs. BMDMs transfected with Scramble and lentiviral vector.
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Fig. 7. miR-155 targets SOCS-1 to regulate inflammatory response in macrophages. A: wild-type 3=-UTR of mouse SOCS-1 (SOCS-1 3=-UTR) or miR-155 binding deficient mutant 3=-UTR of mouse SOCS-1 (mutSOCS-1 3=-UTR) was separately inserted into the pMIR-REPORT miRNA expression reporter vector. Then these vectors were combined with either miR-155 mimics mmu-miR-155-5p miRNA precursor (Pre-miR-155) or miRNA precursor negative control (Scramble) to be cotransfected into HEK-293T cells. The luciferase activity of the reporter vector was assayed, and the relative activity was compared between groups. n 3 samples per group. *P 0.05 vs. control miRNA and SOCS-1 3=-UTR cotransfected group. B: BMDMs were transfected with SOCS-1 siRNA or control nonsense siRNA. Protein levels of SOCS-1 in cell lysates after transfection were assayed by <t>Western</t> <t>blot</t> <t>analysis.</t> C and D: BMDMs were differentiated from miR-155/ mice or WT control mice, respectively. Then these cells were transfected with either SOCS-1 siRNA or control nonsense siRNA. Immediately after transfection, BMDMs were treated with LPS (100 ng/ml) for 6 h, and mRNA levels of TNF- (C) and IL-6 (D) were assayed by real-time PCR analysis. n 3 samples per group.*P 0.05 vs. BMDMs from WT mice transfected with control siRNA. #P 0.05 vs. BMDMs from WT mice transfected with SOCS-1 siRNA. E: miR-155/ BMDMs transfected with either Pre-miR-155 or miRNA Scramble Control. Then, these cells were transfected with lentivirus expressing SOCS-1 cDNA tagged with a mutated SOCS-1 3=UTR (mutSCOS-1), which was unable to bind miR-155. An empty lentivirus vector (lentiviral vector) was used as a control. At 72 h after transfection, protein levels of SOCS-1 in transfected cells were assayed by Western blot analysis. F and G: 72 h after BMDMs were transfected with indicated miRNAs and lentivirus, BMDMs were stimulated with LPS for 6 h. mRNA levels of TNF- (F), and IL-6 (G) were assayed by real-time PCR analysis. n 3 samples per group. *P 0.05 vs. BMDMs transfected with Pre-miR-155 and lentiviral vector. #P 0.05 vs. BMDMs transfected with Scramble and mutSOCS-1. §P 0.05 vs. BMDMs transfected with Scramble and lentiviral vector.
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Fig. 7. miR-155 targets SOCS-1 to regulate inflammatory response in macrophages. A: wild-type 3=-UTR of mouse SOCS-1 (SOCS-1 3=-UTR) or miR-155 binding deficient mutant 3=-UTR of mouse SOCS-1 (mutSOCS-1 3=-UTR) was separately inserted into the pMIR-REPORT miRNA expression reporter vector. Then these vectors were combined with either miR-155 mimics mmu-miR-155-5p miRNA precursor (Pre-miR-155) or miRNA precursor negative control (Scramble) to be cotransfected into HEK-293T cells. The luciferase activity of the reporter vector was assayed, and the relative activity was compared between groups. n 3 samples per group. *P 0.05 vs. control miRNA and SOCS-1 3=-UTR cotransfected group. B: BMDMs were transfected with SOCS-1 siRNA or control nonsense siRNA. Protein levels of SOCS-1 in cell lysates after transfection were assayed by <t>Western</t> <t>blot</t> <t>analysis.</t> C and D: BMDMs were differentiated from miR-155/ mice or WT control mice, respectively. Then these cells were transfected with either SOCS-1 siRNA or control nonsense siRNA. Immediately after transfection, BMDMs were treated with LPS (100 ng/ml) for 6 h, and mRNA levels of TNF- (C) and IL-6 (D) were assayed by real-time PCR analysis. n 3 samples per group.*P 0.05 vs. BMDMs from WT mice transfected with control siRNA. #P 0.05 vs. BMDMs from WT mice transfected with SOCS-1 siRNA. E: miR-155/ BMDMs transfected with either Pre-miR-155 or miRNA Scramble Control. Then, these cells were transfected with lentivirus expressing SOCS-1 cDNA tagged with a mutated SOCS-1 3=UTR (mutSCOS-1), which was unable to bind miR-155. An empty lentivirus vector (lentiviral vector) was used as a control. At 72 h after transfection, protein levels of SOCS-1 in transfected cells were assayed by Western blot analysis. F and G: 72 h after BMDMs were transfected with indicated miRNAs and lentivirus, BMDMs were stimulated with LPS for 6 h. mRNA levels of TNF- (F), and IL-6 (G) were assayed by real-time PCR analysis. n 3 samples per group. *P 0.05 vs. BMDMs transfected with Pre-miR-155 and lentiviral vector. #P 0.05 vs. BMDMs transfected with Scramble and mutSOCS-1. §P 0.05 vs. BMDMs transfected with Scramble and lentiviral vector.
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Fig. 7. miR-155 targets SOCS-1 to regulate inflammatory response in macrophages. A: wild-type 3=-UTR of mouse SOCS-1 (SOCS-1 3=-UTR) or miR-155 binding deficient mutant 3=-UTR of mouse SOCS-1 (mutSOCS-1 3=-UTR) was separately inserted into the pMIR-REPORT miRNA expression reporter vector. Then these vectors were combined with either miR-155 mimics mmu-miR-155-5p miRNA precursor (Pre-miR-155) or miRNA precursor negative control (Scramble) to be cotransfected into HEK-293T cells. The luciferase activity of the reporter vector was assayed, and the relative activity was compared between groups. n 3 samples per group. *P 0.05 vs. control miRNA and SOCS-1 3=-UTR cotransfected group. B: BMDMs were transfected with SOCS-1 siRNA or control nonsense siRNA. Protein levels of SOCS-1 in cell lysates after transfection were assayed by <t>Western</t> <t>blot</t> <t>analysis.</t> C and D: BMDMs were differentiated from miR-155/ mice or WT control mice, respectively. Then these cells were transfected with either SOCS-1 siRNA or control nonsense siRNA. Immediately after transfection, BMDMs were treated with LPS (100 ng/ml) for 6 h, and mRNA levels of TNF- (C) and IL-6 (D) were assayed by real-time PCR analysis. n 3 samples per group.*P 0.05 vs. BMDMs from WT mice transfected with control siRNA. #P 0.05 vs. BMDMs from WT mice transfected with SOCS-1 siRNA. E: miR-155/ BMDMs transfected with either Pre-miR-155 or miRNA Scramble Control. Then, these cells were transfected with lentivirus expressing SOCS-1 cDNA tagged with a mutated SOCS-1 3=UTR (mutSCOS-1), which was unable to bind miR-155. An empty lentivirus vector (lentiviral vector) was used as a control. At 72 h after transfection, protein levels of SOCS-1 in transfected cells were assayed by Western blot analysis. F and G: 72 h after BMDMs were transfected with indicated miRNAs and lentivirus, BMDMs were stimulated with LPS for 6 h. mRNA levels of TNF- (F), and IL-6 (G) were assayed by real-time PCR analysis. n 3 samples per group. *P 0.05 vs. BMDMs transfected with Pre-miR-155 and lentiviral vector. #P 0.05 vs. BMDMs transfected with Scramble and mutSOCS-1. §P 0.05 vs. BMDMs transfected with Scramble and lentiviral vector.
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Fig. 7. miR-155 targets SOCS-1 to regulate inflammatory response in macrophages. A: wild-type 3=-UTR of mouse SOCS-1 (SOCS-1 3=-UTR) or miR-155 binding deficient mutant 3=-UTR of mouse SOCS-1 (mutSOCS-1 3=-UTR) was separately inserted into the pMIR-REPORT miRNA expression reporter vector. Then these vectors were combined with either miR-155 mimics mmu-miR-155-5p miRNA precursor (Pre-miR-155) or miRNA precursor negative control (Scramble) to be cotransfected into HEK-293T cells. The luciferase activity of the reporter vector was assayed, and the relative activity was compared between groups. n 3 samples per group. *P 0.05 vs. control miRNA and SOCS-1 3=-UTR cotransfected group. B: BMDMs were transfected with SOCS-1 siRNA or control nonsense siRNA. Protein levels of SOCS-1 in cell lysates after transfection were assayed by <t>Western</t> <t>blot</t> <t>analysis.</t> C and D: BMDMs were differentiated from miR-155/ mice or WT control mice, respectively. Then these cells were transfected with either SOCS-1 siRNA or control nonsense siRNA. Immediately after transfection, BMDMs were treated with LPS (100 ng/ml) for 6 h, and mRNA levels of TNF- (C) and IL-6 (D) were assayed by real-time PCR analysis. n 3 samples per group.*P 0.05 vs. BMDMs from WT mice transfected with control siRNA. #P 0.05 vs. BMDMs from WT mice transfected with SOCS-1 siRNA. E: miR-155/ BMDMs transfected with either Pre-miR-155 or miRNA Scramble Control. Then, these cells were transfected with lentivirus expressing SOCS-1 cDNA tagged with a mutated SOCS-1 3=UTR (mutSCOS-1), which was unable to bind miR-155. An empty lentivirus vector (lentiviral vector) was used as a control. At 72 h after transfection, protein levels of SOCS-1 in transfected cells were assayed by Western blot analysis. F and G: 72 h after BMDMs were transfected with indicated miRNAs and lentivirus, BMDMs were stimulated with LPS for 6 h. mRNA levels of TNF- (F), and IL-6 (G) were assayed by real-time PCR analysis. n 3 samples per group. *P 0.05 vs. BMDMs transfected with Pre-miR-155 and lentiviral vector. #P 0.05 vs. BMDMs transfected with Scramble and mutSOCS-1. §P 0.05 vs. BMDMs transfected with Scramble and lentiviral vector.
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Fig. 7. miR-155 targets SOCS-1 to regulate inflammatory response in macrophages. A: wild-type 3=-UTR of mouse SOCS-1 (SOCS-1 3=-UTR) or miR-155 binding deficient mutant 3=-UTR of mouse SOCS-1 (mutSOCS-1 3=-UTR) was separately inserted into the pMIR-REPORT miRNA expression reporter vector. Then these vectors were combined with either miR-155 mimics mmu-miR-155-5p miRNA precursor (Pre-miR-155) or miRNA precursor negative control (Scramble) to be cotransfected into HEK-293T cells. The luciferase activity of the reporter vector was assayed, and the relative activity was compared between groups. n 3 samples per group. *P 0.05 vs. control miRNA and SOCS-1 3=-UTR cotransfected group. B: BMDMs were transfected with SOCS-1 siRNA or control nonsense siRNA. Protein levels of SOCS-1 in cell lysates after transfection were assayed by Western blot analysis. C and D: BMDMs were differentiated from miR-155/ mice or WT control mice, respectively. Then these cells were transfected with either SOCS-1 siRNA or control nonsense siRNA. Immediately after transfection, BMDMs were treated with LPS (100 ng/ml) for 6 h, and mRNA levels of TNF- (C) and IL-6 (D) were assayed by real-time PCR analysis. n 3 samples per group.*P 0.05 vs. BMDMs from WT mice transfected with control siRNA. #P 0.05 vs. BMDMs from WT mice transfected with SOCS-1 siRNA. E: miR-155/ BMDMs transfected with either Pre-miR-155 or miRNA Scramble Control. Then, these cells were transfected with lentivirus expressing SOCS-1 cDNA tagged with a mutated SOCS-1 3=UTR (mutSCOS-1), which was unable to bind miR-155. An empty lentivirus vector (lentiviral vector) was used as a control. At 72 h after transfection, protein levels of SOCS-1 in transfected cells were assayed by Western blot analysis. F and G: 72 h after BMDMs were transfected with indicated miRNAs and lentivirus, BMDMs were stimulated with LPS for 6 h. mRNA levels of TNF- (F), and IL-6 (G) were assayed by real-time PCR analysis. n 3 samples per group. *P 0.05 vs. BMDMs transfected with Pre-miR-155 and lentiviral vector. #P 0.05 vs. BMDMs transfected with Scramble and mutSOCS-1. §P 0.05 vs. BMDMs transfected with Scramble and lentiviral vector.

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Macrophage micro-RNA-155 promotes lipopolysaccharide-induced acute lung injury in mice and rats.

doi: 10.1152/ajplung.00001.2016

Figure Lengend Snippet: Fig. 7. miR-155 targets SOCS-1 to regulate inflammatory response in macrophages. A: wild-type 3=-UTR of mouse SOCS-1 (SOCS-1 3=-UTR) or miR-155 binding deficient mutant 3=-UTR of mouse SOCS-1 (mutSOCS-1 3=-UTR) was separately inserted into the pMIR-REPORT miRNA expression reporter vector. Then these vectors were combined with either miR-155 mimics mmu-miR-155-5p miRNA precursor (Pre-miR-155) or miRNA precursor negative control (Scramble) to be cotransfected into HEK-293T cells. The luciferase activity of the reporter vector was assayed, and the relative activity was compared between groups. n 3 samples per group. *P 0.05 vs. control miRNA and SOCS-1 3=-UTR cotransfected group. B: BMDMs were transfected with SOCS-1 siRNA or control nonsense siRNA. Protein levels of SOCS-1 in cell lysates after transfection were assayed by Western blot analysis. C and D: BMDMs were differentiated from miR-155/ mice or WT control mice, respectively. Then these cells were transfected with either SOCS-1 siRNA or control nonsense siRNA. Immediately after transfection, BMDMs were treated with LPS (100 ng/ml) for 6 h, and mRNA levels of TNF- (C) and IL-6 (D) were assayed by real-time PCR analysis. n 3 samples per group.*P 0.05 vs. BMDMs from WT mice transfected with control siRNA. #P 0.05 vs. BMDMs from WT mice transfected with SOCS-1 siRNA. E: miR-155/ BMDMs transfected with either Pre-miR-155 or miRNA Scramble Control. Then, these cells were transfected with lentivirus expressing SOCS-1 cDNA tagged with a mutated SOCS-1 3=UTR (mutSCOS-1), which was unable to bind miR-155. An empty lentivirus vector (lentiviral vector) was used as a control. At 72 h after transfection, protein levels of SOCS-1 in transfected cells were assayed by Western blot analysis. F and G: 72 h after BMDMs were transfected with indicated miRNAs and lentivirus, BMDMs were stimulated with LPS for 6 h. mRNA levels of TNF- (F), and IL-6 (G) were assayed by real-time PCR analysis. n 3 samples per group. *P 0.05 vs. BMDMs transfected with Pre-miR-155 and lentiviral vector. #P 0.05 vs. BMDMs transfected with Scramble and mutSOCS-1. §P 0.05 vs. BMDMs transfected with Scramble and lentiviral vector.

Article Snippet: The following antibodies were used as primary antibodies for Western blot analysis: anti-p-65 (4764; Cell Signaling Technology, Danvers, MA); anti-p-p65 (3033; Cell Signaling Technology); anti-I B (4812; Cell Signaling Technology); antip-I B (2859; Cell Signaling Technology); anti-JNK (9258; Cell Signaling Technology); anti-p-JNK (4668; Cell Signaling Technology); anti-P38 (9212; Cell Signaling Technology); anti-p-P38 (4511; Cell Signaling Technology); anti-SHIP-1 (ab59338; Abcam); antiBcl-6 (ab86861; Abcam); anti-SOCS-1 (ab9870; Abcam).

Techniques: Binding Assay, Mutagenesis, Expressing, Plasmid Preparation, Negative Control, Luciferase, Activity Assay, Control, Transfection, Western Blot, Real-time Polymerase Chain Reaction