ago2  (MedChemExpress)

Journal: Frontiers in Pharmacology

Article Title: miR-21-5p attenuates hyperoxia-induced lung injury by modulating YAP1-dependent ferroptosis

doi: 10.3389/fphar.2026.1804152

Figure Lengend Snippet: miR-21-5p directly targets and suppresses YAP1 expression in alveolar epithelial cells (AECs) (A) Relative expression of miR-21-5p in cells transfected with miR-21-5p mimic, inhibitor, or corresponding negative controls (NC), as determined by quantitative real-time polymerase chain reaction (RT-qPCR). (B) Relative mRNA expression of YAP1 following miR-21-5p modulation, measured by RT-qPCR. (C) Western blot analysis and quantification of YAP1 protein expression following miR-21-5p modulation. (D) Schematic representation of the predicted binding site between miR-21-5p and the 3′untranslated region (3′UTR) of YAP1 mRNA, including wild-type (WT) and mutant (MUT) constructs. (E) Dual-luciferase reporter assay showing the interaction between miR-21-5p and YAP1 3′UTR. (F) RNA immunoprecipitation (RIP) assay was performed using an anti-Ago2 antibody, with IgG as a negative control, to validate the interaction between miR-21-5p and YAP1. Data are presented as mean ± SD from six independent biological replicates (n = 6). Statistical analysis was performed as described in the Materials and Methods. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Primary antibodies included anti-YAP1 (CST, #14074, 1:1000), anti-ACSL4 (HUABIO, ET7111-43, 1:2000), anti-SLC7A11 (HUABIO, HA721868, 1:1000), anti-GPX4 (HUABIO, ET1706-45, 1:2000), anti-Ago2 (HUABIO, ET1702-39, 1:2000), and anti-β-actin (HUABIO, M1210-2, 1:20000).

Techniques: Expressing, Transfection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Binding Assay, Mutagenesis, Construct, Luciferase, Reporter Assay, RNA Immunoprecipitation, Negative Control

Journal: iScience

Article Title: miR-181d coordinates homologous recombination and anti-tumor immune responses in glioblastoma

doi: 10.1016/j.isci.2026.115077

Figure Lengend Snippet: miR-181d binds to RAD51 3′ UTR and regulates RAD51 expression (A) Predicted miR-181d MRE and mutated miR-181d MRE within 3′ UTRs of RAD51 . (B) Empty vector or constructs bearing the full length 3′ UTR or mut-3′ UTR fragment of RAD51 were co-transfected with miR-181d mimic into A1207 cells. Luciferase activities were measured 48 h post-transfection. ∗∗ p < 0.01 (Student’s t test) compared to corresponding luciferase reporter bearing miR-181d MRE. Data are presented as the mean ± SD ( n = 3 independent experiments). (C) LN340 cells were transfected with Myc-FLAG -tagged RAD51 cDNA construct with or without miR-181d MRE or mutated MRE (disrupting miR-181d binding) at 3′ UTR. Twenty-four hours after transfection, total RNA was extracted, and RAD51 mRNA expression was analyzed by RT-qPCR, using primers specific to the endogenous (endo)-RAD51 or Myc-FLAG-RAD51 . (D) CMK3 cells expressing Myc-FLAG-RAD51 were transfected with biotinylated (Bi)-miR181d or Bi-miR-NT. The lysate was affinity purified with streptavidin-coated magnetic beads. Isolated RNA was analyzed for endo-RAD51 or Myc-FLAG-RAD51 mRNAs. ∗∗∗ p < 0.001. (E) LN340 cells expressing the Myc-FLAG-RAD51 cDNA construct containing MRE or mut-MRE were transfected with Bi-miR-181d mimic. The lysate was affinity purified with streptavidin-coated magnetic beads. Isolated RNA was analyzed for Myc-FLAG-RAD51 transcripts. Data are represented as the mean ± SD. ∗∗∗ p < 0.001. (F) Schematic of the experimental workflow of Argonaut 2 (AGO2) complex immunoprecipitation. (G) LN340 cells were transfected with Bi-NT or Bi-miR-181d. Bi-miRs were affinity purified by streptavidin pull-down from the cell lysate. The streptavidin pull-down fractions were incubated with free biotin (4 mg/mL) for competitive elution of AGO2-bound complex and subjected to western blot analysis. The eluates underwent AGO2 complex immunoprecipitation followed by RT-qPCR for RAD51 transcripts. The represented RAD51 transcript copy numbers are normalized to IgG of corresponding samples.

Article Snippet: Anti-AGO2 antibody , Proteintech , RRID: AB_2918686.

Techniques: Expressing, Plasmid Preparation, Construct, Transfection, Luciferase, Binding Assay, Quantitative RT-PCR, Affinity Purification, Magnetic Beads, Isolation, Immunoprecipitation, Incubation, Western Blot