mutant probe  (Macrogen)

Journal: Molecular Medicine Reports

Article Title: circRNA hsa_circ_0072107 aggravates myocardial hypertrophy via its function as a competitive endogenous RNA of miR-516b-5p

doi: 10.3892/mmr.2025.13597

Figure Lengend Snippet: miR-516b-5p is a miRNA binding to hsa_circ_0072107. (A) Venn diagram showed that two miRNAs may bind to hsa_circ_0072107. (B) The binding site information of miR-516b-5p and miR-510-5p on hsa_circ_0072107. (C) Reverse transcription-quantitative PCR validation of transfection efficiency in cells transfected with miR-NC, miR-516b-5p mimics and inhibitors. (D) hsa_circ_0072107, miR-516b-5p and miR-510-5p levels in RNA enriched by circ_0072107 probe or scramble probe during biotinylated probe pull-down assay. (E) hsa_circ_0072107 level in miRNA complexes enriched by anti-AGO or IgG during anti-AGO2 RIP assay. AC16 cells transfected with miR-516b-5p mimic or miR-NC were used. (F) The effect of miR-516b-5p mimic or miR-NC on the relative luciferase activity of luciferase reporter with linear circ_0072107 or linear mutcirc_0072107 as the artificial 3′ UTR of Renilla , respectively. *P<0.05, **P<0.01, n=3. miRNA, microRNA; AGO2, argonaute 2; RIP, RNA immunoprecipitation; NC, negative control.

Article Snippet: Biotin-labelled miR-516b-5p probe (5′-bio-AUCUGGAGGUAAGAAGCACUUU-3′) or biotin-labelled mutant miR-516b-5p probe (5′-bio-ACUCAAGAGUAAGAAGCACUUU-3′; Suzhou GenePharma Co., Ltd.) were incubated with cell lysates, and the pull-down assay was carried out using the Pierce Magnetic RNA-Protein Pull-Down Kit (Pierce; Thermo Fisher Scientific, Inc.).

Techniques: Binding Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Biomarker Discovery, Transfection, Pull Down Assay, Luciferase, Activity Assay, RNA Immunoprecipitation, Negative Control

Journal: Molecular Medicine Reports

Article Title: circRNA hsa_circ_0072107 aggravates myocardial hypertrophy via its function as a competitive endogenous RNA of miR-516b-5p

doi: 10.3892/mmr.2025.13597

Figure Lengend Snippet: Overexpression of hsa_circ_0072107 with mutant miR-516b-5p binding site cannot induce AC16 hypertrophy. (A) miR-516b-5p expression level in AC16 cells transfected with empty vector (vector), hsa_circ_0072107 overexpression vector (ov-circ_0072107) and overexpression vector of hsa_circ_0072107 with mutant miR-516b-5p binding site (mutov-circ_0072107). No significant difference was observed in (B) cell size (scale bars, 50 µm), (C) protein/DNA ratio, or (D) levels of BNP and β-MHC between the vector and mutov-circ_0072107 groups. miRNA, microRNA.

Article Snippet: Biotin-labelled miR-516b-5p probe (5′-bio-AUCUGGAGGUAAGAAGCACUUU-3′) or biotin-labelled mutant miR-516b-5p probe (5′-bio-ACUCAAGAGUAAGAAGCACUUU-3′; Suzhou GenePharma Co., Ltd.) were incubated with cell lysates, and the pull-down assay was carried out using the Pierce Magnetic RNA-Protein Pull-Down Kit (Pierce; Thermo Fisher Scientific, Inc.).

Techniques: Over Expression, Mutagenesis, Binding Assay, Expressing, Transfection, Plasmid Preparation

Journal: Molecular Medicine Reports

Article Title: circRNA hsa_circ_0072107 aggravates myocardial hypertrophy via its function as a competitive endogenous RNA of miR-516b-5p

doi: 10.3892/mmr.2025.13597

Figure Lengend Snippet: ZFP36 is a target gene of miR-516b-5p. (A) Venn diagram identified differentially expressed target genes of miR-516b-5p. In the GSE32453 dataset, 461 differentially expressed mRNAs were identified in the myocardium of patients with hypertrophic cardiomyopathy. (B) The binding sites of miR-516b-5p on ZFP36 3′UTR. (C) ZFP36 mRNA level in RNA enriched by miR-516b-5p probe or mutant miR-516b-5p probe in a biotinylated probe pull-down assay. (D) The effect of miR-NC or miR-516b-5p mimic on the relative luciferase activity of luciferase reporter using ZFP36 3′UTR or mutant ZFP36 3′UTR (mutZFP36 3′UTR) as the artificial 3′ UTR of Renilla . (E) The protein level of ZFP36 in AC16 cells transfected with miR-NC, miR-516b-5p, miR-NC inhibitor, or miR-516b-5p inhibitor. *P<0.05, n=3. ZFP36, zinc ring finger protein 36; miRNA, microRNA; NC, negative control.

Article Snippet: Biotin-labelled miR-516b-5p probe (5′-bio-AUCUGGAGGUAAGAAGCACUUU-3′) or biotin-labelled mutant miR-516b-5p probe (5′-bio-ACUCAAGAGUAAGAAGCACUUU-3′; Suzhou GenePharma Co., Ltd.) were incubated with cell lysates, and the pull-down assay was carried out using the Pierce Magnetic RNA-Protein Pull-Down Kit (Pierce; Thermo Fisher Scientific, Inc.).

Techniques: Binding Assay, Mutagenesis, Pull Down Assay, Luciferase, Activity Assay, Transfection, Negative Control

Journal: Molecular Medicine Reports

Article Title: circRNA hsa_circ_0072107 aggravates myocardial hypertrophy via its function as a competitive endogenous RNA of miR-516b-5p

doi: 10.3892/mmr.2025.13597

Figure Lengend Snippet: Effect of miR-516b-5p overexpression on AC16 hypertrophy and ZFP36 expression was blocked by hsa_circ_0072107 overexpression. Under ISO treatment, hsa_circ_0072107 overexpression vector (ov-circ_0072107), miR-516b-5p inhibitor, miR-516b-5p mimic (miR-516b-5p), or miR-516b-5p mimic plus ov-circ_0072107 was transfected into AC16 cells, respectively and the transfection reagent was used as negative control (ISO + NC). (A) miR-516b-5p expression level effects on (B) cell size (scale bars, 50 µm), (C) protein/DNA ratio and (D) the protein levels of BNP and β-MHC were measured. (E and F) The protein level of ZFP36 in different groups was detected by western blotting. (G) Relative ZFP36 levels measured by reverse transcription-quantitative PCR in different experimental groups. (H) Western blot analysis of ZFP36 protein expression in different experimental groups, with quantification of protein expression levels shown in the histogram. *P<0.05, n=3. miRNA, microRNA; ZFP36, zinc ring finger protein 36; ISO, isoproterenol; NC, negative control; vector group, cells transfected with empty vector; ov-circ_0072107, cells transfected with hsa_circ_0072107 overexpression vector; si-NC group, cells transfected with negative siRNA; si-circ_0072107 group, cells transfected with siRNA targeting hsa_circ_0072107 (si-circ_0072107).

Article Snippet: Biotin-labelled miR-516b-5p probe (5′-bio-AUCUGGAGGUAAGAAGCACUUU-3′) or biotin-labelled mutant miR-516b-5p probe (5′-bio-ACUCAAGAGUAAGAAGCACUUU-3′; Suzhou GenePharma Co., Ltd.) were incubated with cell lysates, and the pull-down assay was carried out using the Pierce Magnetic RNA-Protein Pull-Down Kit (Pierce; Thermo Fisher Scientific, Inc.).

Techniques: Over Expression, Expressing, Plasmid Preparation, Transfection, Negative Control, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Nature Communications

Article Title: Two-component system GrpP/GrpQ promotes pathogenicity of uropathogenic Escherichia coli CFT073 by upregulating type 1 fimbria

doi: 10.1038/s41467-025-55982-z

Figure Lengend Snippet: A Competitive EMSAs of the binding of the fimS DNA fragment with phosphorylated GrpQ protein Kana fragment was used as the negative control. Representative images from three independent experiments. B Fold changes of the fimS DNA fragment in the GrpQ-ChIP sample relative to the mock-ChIP sample. Data were obtained from three independent experiments and presented as mean ± SD. P values were determined using a two-way analysis of variance. C DNase I footprinting assay identified precise GrpQ binding sites on fimS . The protected region shows a significantly reduced peak intensity (blue) pattern compared with that of the control (red). D Competitive EMSAs of the binding of purified phosphorylated GrpQ to the mutant fimS binding region. Representative images from three independent experiments. E Quantification of the percentage of bacteria with fimS in the phase-OFF orientation. F Phase-OFF orientation of fimS was calculated using qPCR in Δ grpP/grpQ relative to WT. Data were obtained from three independent experiments and presented as mean ± SD ( B , E, and F ). P values were determined using two-way analysis of variance ( B ) or two-tailed Student’s t -test ( E and F ). Significance was indicated by a P value. n.s., No significant difference. B P = 0.00004; F P = 0.000099. Source data are provided as a Source data file.

Article Snippet: DNA probes of fimS and the mutant fimS (the binding motif was mutated to 5ʹ-CCGTGCGCTGGGTGACCGAGCATCCCCCA-3ʹ) were amplified respectively with and without 6-FAM-labeled primers and purified (QIAGEN; 28006).

Techniques: Binding Assay, Negative Control, Footprinting, Control, Purification, Mutagenesis, Bacteria, Two Tailed Test