Journal: Cell Death & Disease

Article Title: Renal inhibition of miR-181a ameliorates 5-fluorouracil-induced mesangial cell apoptosis and nephrotoxicity

doi: 10.1038/s41419-018-0677-8

Figure Lengend Snippet: a , b Analysis of miR-181a ( a ) and BIRC ( b ) in mesangial cells treated with 5-FU (100 μM) for 6, 12, 24, or 48 h. ** p < 0.01 vs. control, n = 6. c , d miR-181a expression was inversely correlated with BIRC6 mRNA expression ( c ) and positively correlated with apoptotic rate ( d ). e The cells were pretreated with miR-181a mimics or miR-181a inhibitor for 48 h followed by incubation of 5-FU (100 μM) for another 24 h. Cell morphology was assessed by phase contrast microscopy. f Cell viability was assessed by CCK-8 assay. ** p < 0.01 vs. control; ## p < 0.01 vs. 5-FU, n = 6. g Hoechst 33258 nuclei staining was used to detect apoptotic morphology. h Fluorescence-activated cell sorting-derived dot plot diagrams of mesangial cells stained with annexin V-APC/7-AAD. i Percentage of apoptotic cells was determined by quantitative analysis. ** p < 0.01 vs. control; ## p < 0.01 vs. 5-FU, n = 4

Article Snippet: The miR-181a tough decoy (TuD) sequences (5′-ACTCACCGACAGCATCTGTTGAATGTT-3′) were synthesized and cloned into pDKD-CMV-eGFP-U6 plasmid by GenScript.

Techniques: Control, Expressing, Incubation, Microscopy, CCK-8 Assay, Staining, Fluorescence, FACS, Derivative Assay

Journal: Cell Death & Disease

Article Title: Renal inhibition of miR-181a ameliorates 5-fluorouracil-induced mesangial cell apoptosis and nephrotoxicity

doi: 10.1038/s41419-018-0677-8

Figure Lengend Snippet: a – c Cells were pretreated with miR-181a mimics or miR-181a inhibitor for 48 h prior to incubation of 5-FU (100 μM) for another 24 h. The protein expression of p53 ( a ), Bcl-2 ( b ), and Bax ( c ) were determined by western blotting. d Mitochondrial membrane potential was measured by JC-1 staining. e Quantitative analysis of the green/red fluorescence intensity ratio, which represented as a surrogate marker of mitochondrial membrane depolarization. f Western blotting analyses of 5-FU-induced mitochondrial cytochrome c (Cyto c) release from mitochondria into the cytosol. g , h Caspase-9 ( g ) and caspase-3 ( h ) cleavage were detected by western blotting. ** p < 0.01 vs. control; ## p < 0.01 vs. 5-FU, n = 6

Article Snippet: The miR-181a tough decoy (TuD) sequences (5′-ACTCACCGACAGCATCTGTTGAATGTT-3′) were synthesized and cloned into pDKD-CMV-eGFP-U6 plasmid by GenScript.

Techniques: Incubation, Expressing, Western Blot, Membrane, Staining, Fluorescence, Marker, Control

Journal: Cell Death & Disease

Article Title: Renal inhibition of miR-181a ameliorates 5-fluorouracil-induced mesangial cell apoptosis and nephrotoxicity

doi: 10.1038/s41419-018-0677-8

Figure Lengend Snippet: a , b Cells were transfected with miR-181a mimics or miR-181a inhibitor for 48 h. The protein ( a ) and mRNA ( b ) expression of p53 were determined by western blotting and qRT-PCR, respectively. ** p < 0.01 vs. miRNA negative control or miRNA inhibitor negative control, n = 5. c , d Mesangial cells pretreated with miR-181a mimics ( c ) or miR-181a inhibitor ( d ) were incubated with cycloheximide (CHX, 10 μg/ml) for indicated time period. Representative western blotting image of p53 and relative intensity of p53 protein expression were shown. e Cells were treated with or without MG-132 (20 μM) for 12 h prior to transfection with miRNA inhibitor negative control or miR-181a inhibitor for another 48 h. P53 protein expression was analyzed by western blotting. ** p < 0.01 vs. miRNA inhibitor negative control; ## p < 0.01 vs. miR-181a inhibitor, n = 6. f Cell lysates were immunoprecipitated with p53 antibody and the ubiquitination of p53 was detected by western blotting using ubiquitin antibody. g HCT116 p53 +/+ cells and HCT116 p53 −/− cells were co-transfected with miR-181a mimics or negative control and Bax luciferase (Bax-Luc) which contains the binding site of p53 for luciferase activity assay. h The mRNA expression of p53 target genes, including Bax, p21, Puma and Fas, was detected in HCT116 p53 +/+ cells and HCT116 p53 −/− cells after miR-181a mimics transfection. ** p < 0.01 vs. miRNA negative control, n = 6

Article Snippet: The miR-181a tough decoy (TuD) sequences (5′-ACTCACCGACAGCATCTGTTGAATGTT-3′) were synthesized and cloned into pDKD-CMV-eGFP-U6 plasmid by GenScript.

Techniques: Transfection, Expressing, Western Blot, Quantitative RT-PCR, Negative Control, Incubation, Immunoprecipitation, Ubiquitin Proteomics, Luciferase, Binding Assay, Activity Assay

Journal: Cell Death & Disease

Article Title: Renal inhibition of miR-181a ameliorates 5-fluorouracil-induced mesangial cell apoptosis and nephrotoxicity

doi: 10.1038/s41419-018-0677-8

Figure Lengend Snippet: a The schematic of the luciferase reporter containing the binding sites for miR-181a at the 3′-untranslated regions (3′-UTR) of BIRC6 from different species. The sequences of BIRC6 3′-UTR mutants used for luciferase reporter assay are presented. b , c Luciferase reporter constructs containing 3′-UTR ( b ) or mutant one 3′-UTR (3′-UTR mutant 1) or mutant double 3′-UTR (3′-UTR double mutant) ( c ) of BIRC6 was co-transfected with miRNA negative control (NC-m), miR-181a mimics (miR-181a-m), miRNA inhibitor negative control (NC-i), or miR-181a inhibitor (miR-181a-i) in mesangial cells and the luciferase activity were determined. d , e Mesangial cells were transfected with miR-181a mimics or miR-181a inhibitor for 48 h. qRT-PCR and western blotting were performed to examine endogenous BIRC6 mRNA ( d ) and protein ( e ) expression. ** p < 0.01 vs. miRNA negative control or miRNA inhibitor negative control, n = 6

Article Snippet: The miR-181a tough decoy (TuD) sequences (5′-ACTCACCGACAGCATCTGTTGAATGTT-3′) were synthesized and cloned into pDKD-CMV-eGFP-U6 plasmid by GenScript.

Techniques: Luciferase, Binding Assay, Reporter Assay, Construct, Mutagenesis, Transfection, Negative Control, Activity Assay, Quantitative RT-PCR, Western Blot, Expressing

Journal: Cell Death & Disease

Article Title: Renal inhibition of miR-181a ameliorates 5-fluorouracil-induced mesangial cell apoptosis and nephrotoxicity

doi: 10.1038/s41419-018-0677-8

Figure Lengend Snippet: a Experimental design to examine the effect of miR-181a inhibition on 5-FU-induced kidney injury. Mice were injected with adeno-associated virus encoding miR-181a tough decoy (AAV-TuD-181a) or AAV-TuD-GFP for 14 days and then treated with 5-FU (200 mg/kg body weight) for 4 days. b On the 18th day, mice were sacrificed, and the miR-181a expression in kidneys was determined. c – e Serum blood urea nitrogen (BUN) ( c ), creatinine ( d ), and LDH ( e ) concentration were tested by biochemical assay. ** p < 0.01 vs. control; ## p < 0.01 vs. 5-FU, n = 5–10. f Histological examination of renal cortex section was performed using hematoxylin & eosin (H&E) staining. n = 5

Article Snippet: The miR-181a tough decoy (TuD) sequences (5′-ACTCACCGACAGCATCTGTTGAATGTT-3′) were synthesized and cloned into pDKD-CMV-eGFP-U6 plasmid by GenScript.

Techniques: Inhibition, Injection, Virus, Expressing, Concentration Assay, Control, Staining

Journal: Cell Death & Disease

Article Title: Renal inhibition of miR-181a ameliorates 5-fluorouracil-induced mesangial cell apoptosis and nephrotoxicity

doi: 10.1038/s41419-018-0677-8

Figure Lengend Snippet: a , b Immunohistochemical staining of Ly-6G ( a ) and CD68 ( b ) in kidney tissues of mice treated with TuD-GFP or TuD-181a under basal level or after 5-FU administration. c , d Quantification of Ly-6G-positive neutrophils ( c ) and CD68-positive macrophages ( d ) areas using Image-Pro 5.0 software. e The myeloperoxidase (MPO) activity in kidney tissues was determined. f – h The mRNA expressions of IL-1β ( f ), IL-6 ( g ), and TNF-α ( h ) were determined by qRT-PCR, respectively. ** p < 0.01 vs. control; ## p < 0.01 vs. 5-FU, n = 4–8

Article Snippet: The miR-181a tough decoy (TuD) sequences (5′-ACTCACCGACAGCATCTGTTGAATGTT-3′) were synthesized and cloned into pDKD-CMV-eGFP-U6 plasmid by GenScript.

Techniques: Immunohistochemical staining, Staining, Software, Activity Assay, Quantitative RT-PCR, Control

Journal: Cell Death & Disease

Article Title: Renal inhibition of miR-181a ameliorates 5-fluorouracil-induced mesangial cell apoptosis and nephrotoxicity

doi: 10.1038/s41419-018-0677-8

Figure Lengend Snippet: a – g Histological analysis of renal cortex section from TuD-GFP- or TuD-181a-treated mice under basal level or after 5-FU administration, including TUNEL ( a ), BIRC6 ( b ), p53 ( c ), Bax ( d ), Bcl-2 ( e ), cleaved caspase-9 ( f ), and cleaved caspase-3 ( g ) staining. Representative images were shown from six independent experiments

Article Snippet: The miR-181a tough decoy (TuD) sequences (5′-ACTCACCGACAGCATCTGTTGAATGTT-3′) were synthesized and cloned into pDKD-CMV-eGFP-U6 plasmid by GenScript.

Techniques: TUNEL Assay, Staining