Journal: Thoracic Cancer

Article Title: Identification of a novel circ_0001946/ miR ‐1290/ SOX6 ceRNA network in esophageal squamous cell cancer

doi: 10.1111/1759-7714.14381

Figure Lengend Snippet: Circ_0001946 binds to miR‐1290. (a) Schematic representation of the miR‐1290 target sequence within circ_0001946. The predicted miR‐1290 seed region was mutated. (b) The firefly luciferase reporter constructs with the wild‐type (circ_0001946 wt) or mutant‐type (circ_0001946 mut) miR‐1290 seed region were cotransfected in 293 T cells along with pRL‐TK Renilla luciferase vector and miR‐1290 mimic or mimic mock. Cotransfection of miR‐1290 mimic repressed the luciferase activity of circ_0001946 wt not circ_0001946 mut. (c) Lysates were incubated with anti‐IgG or anti‐Ago2 antibody and magnetic beads. qRT‐PCR showing the enrichment levels of circ_0001946 and miR‐1290 in Ago2‐associated RNA. (d) qRT‐PCR showing the overexpression of miR‐1290 in 77 ESCC tissue specimens compared with the adjacent nontumor esophageal tissues from the same patients. (e) Scatter plots of miR‐1290 expression versus circ_0001946 level in 77 ESCC tissue specimens. Pearson's correlation coefficients (r) and p ‐values are shown. (f) qRT‐PCR showing the elevation of circ_0001946 expression in ECA109 and KYSE450 ESCC cells compared to the nontumor HET‐1A cells. * p < 0.05

Article Snippet: We incubated cell lysates prepared in RIPA with rabbit Argonaute2 (Ago2) monoclonal antibody (ab186733, 1:30; Abcam) or isotype IgG control (ab172730, 1:100, Abcam) for 3 h at 4°C before adding magnetic beads (A/G) (MedChemExpress) for an additional 3 h. We extracted Ago2‐associated RNA from the beads to evaluate circ_0001946 and miR‐1290 levels using qRT‐PCR.

Techniques: Sequencing, Luciferase, Construct, Mutagenesis, Plasmid Preparation, Cotransfection, Activity Assay, Incubation, Magnetic Beads, Quantitative RT-PCR, Over Expression, Expressing

Journal: Molecular human reproduction

Article Title: The expression of Argonaute2 and related microRNA biogenesis proteins in normal and hypoxic trophoblasts.

doi: 10.1093/molehr/gam006

Figure Lengend Snippet: Figure 4. The expression of Argonaute2 (Ago2) and DP103 proteins in hypoxic trophoblasts. The expression of Ago2 (A) and DP103 (B) in tropho- blasts. Protein expression was determined by western blot (left panels) in tro- phoblasts cultured for 72 h in standard (S) or hypoxic (H) conditions. The results were quantified using densitometry, with expression at 24 h in standard conditions, defined as one. None of the differences were statistically significant. Note that a non-specific band was detected below the Ago2 band in all para- digms. This band has been described by the manufacturer and disappeared by immunoprecipitation (Figure 5). Protein expression was also determined by immunofluorescence (right panels) of placental villi from normal (1–3) or fetal growth restriction (FGR) (4–6) placentas from dizygotic twins (represen- tative experiments of three pairs of twins). Panels 1 and 4 depict staining for Ago2 or DP103 (green) with TOPRO-3 staining (blue) for nuclei. Panels 2 and 5 are a higher magnification for Ago2 or DP103, and panels 3 and 6 are controls in which the primary antibody was omitted (bar ¼ 20 mm).

Article Snippet: The modified calcium–phosphate co-precipitation method was used for transfection, as we previously described (Yan et al., 2003), using 3 mg of DNA plasmid expressing human Ago2 or enhanced green fluorescent protein (EGFP) (pIRESneoFLAG/HA-Ago2, pIRESneo-FLAG/HA EGFP, Addgene, Cambridge, MA, USA).

Techniques: Expressing, Western Blot, Cell Culture, Immunoprecipitation, Staining

Journal: Molecular human reproduction

Article Title: The expression of Argonaute2 and related microRNA biogenesis proteins in normal and hypoxic trophoblasts.

doi: 10.1093/molehr/gam006

Figure Lengend Snippet: Figure 6. Ago2 expression level is not a limiting factor in trophoblast response to hypoxia. (A) The influence of hypoxia on vascular endothelial growth factor (VEGF), b-hCG and p53 transcript levels in JEG3 cells. Transcript levels were determined in duplicates as described in materials and methods (representative experiments, n ¼ 3). Expression levels are expressed as fold over expression at 24 h, defined as one. *Denotes P , 0.05. (B) Transient over-transfection of Ago2 effectively increased Ago2 mRNA and protein expression levels in JEG3 cells cultured in standard or hypoxic conditions when compared with transfection with an enhanced green fluorescent protein (EGFP)-expressing plasmid control. Ago2 protein overexpression (right panel) resulted in 2–12-fold overexpression of Ago2 (n ¼ 3). The non-specific band described in Figure 4 was again detected below the Ago2 band. (C) The effect of Ago2 overexpression in JEG3 cells on mRNA levels for VEGF, b-hCG and p53 in standard or hypoxic conditions (n ¼ 3). The expression of each transcript was determined in duplicate and expressed as fold overexpression at 24 h in EGFP control overexpression, defined as one for standard or for hypoxia. The differences were not statistically significant.

Article Snippet: The modified calcium–phosphate co-precipitation method was used for transfection, as we previously described (Yan et al., 2003), using 3 mg of DNA plasmid expressing human Ago2 or enhanced green fluorescent protein (EGFP) (pIRESneoFLAG/HA-Ago2, pIRESneo-FLAG/HA EGFP, Addgene, Cambridge, MA, USA).

Techniques: Expressing, Over Expression, Transfection, Cell Culture, Plasmid Preparation, Control

Journal: Molecular human reproduction

Article Title: The expression of Argonaute2 and related microRNA biogenesis proteins in normal and hypoxic trophoblasts.

doi: 10.1093/molehr/gam006

Figure Lengend Snippet: Figure 5. DP103 forms a complex with Ago2 in trophoblasts cultured in stan- dard or hypoxic conditions. Protein complexes were precipitated using protein-A bead bound anti-Ago2 antibodies and detected using anti-DP103. Co-immunoprecipitation (a representative experiment, n ¼ 3) was performed as described in Materials and methods, with the total amount of protein input indicated below the gel. The lower graph depicts the ratio between DP103 and Ago2 in standard or hypoxic conditions, with band intensity quantified using densitometry.

Article Snippet: The modified calcium–phosphate co-precipitation method was used for transfection, as we previously described (Yan et al., 2003), using 3 mg of DNA plasmid expressing human Ago2 or enhanced green fluorescent protein (EGFP) (pIRESneoFLAG/HA-Ago2, pIRESneo-FLAG/HA EGFP, Addgene, Cambridge, MA, USA).

Techniques: Cell Culture, Immunoprecipitation

Journal: Life Science Alliance

Article Title: miR-486 is essential for muscle function and suppresses a dystrophic transcriptome

doi: 10.26508/lsa.202101215

Figure Lengend Snippet: (A) Schematic demonstrating the workflow for the chimeric eCLIP sequencing platform to identify miR-486 in vivo skeletal muscle regulated transcripts. Tibialis anterior muscles were harvested from 6-mo-old WT and mir-486 KO male mice, and total RNA isolation was completed. The Ago2-miR-486 complex bound to target RNA transcripts was isolated, and then sequencing was performed to map the miR-486–associated peak tracks. The sequence logo (AUGUACAG) represents the consensus sequence for the top 18 miR-486–associated peak reads based on the miRNA:mRNA target sequencing alignments. The miR-486 seed sequence is shown in the 5′–3′ direction. (B) Chromosomal location of a single top hit peak transcript, Mt2 , as identified by eCLIP-seq as a direct target of miR-486. Peaks generated using Integrative Genome Viewer. (C) Metagene plot demonstrating overall miR-486 binding location by a relative position on the target gene. (D) Pie chart demonstrating the proportion of miR-486 gene targets and the respective intragenic binding location of miR-486. (E) Table outlining the top 10 transcripts that were identified as direct targets of miR-486 via CLIP-seq. (F) g:Profiler enrichment analysis graph demonstrates the most significant cellular pathways associated with the 18 direct miR-486 targets identified via chimeric eCLIP-seq. The pathway ID number in the table correlates with the numbered dots in the accompanying graph above. (G) Heat map expression of quantitative PCR of 18 miR-486 eCLIP-seq targets in mir-486 KO and mdx 5cv tibialis anterior muscles expression levels compared with WT controls in separate cohort analyses. Data points are individual biological replicates, N = 4/cohort, and logarithmic fold change normalized to both wild type and β-actin is shown. ** P ≤ 0.01 N = 3 replicates per cohort. Transcript levels are normalized to β-actin, and mir-486 KO levels are shown as relative to WT.

Article Snippet: A total of 10% of IP samples and 1% of input samples were run on NuPAGE 4–12% Bis-Tris protein gels, transferred to PVDF membrane, and probed with 1:1,000 of AGO2 WB antibody and 1:10,000 TrueBlot anti-Rabbit IgG (HRP) (Rockland Immunochemicals) and imaged with a C300 Imager for 1 min on normal settings using Azure Radiance ECL.

Techniques: Sequencing, In Vivo, Muscles, Isolation, Generated, Binding Assay, Expressing, Real-time Polymerase Chain Reaction

Journal: RNA

Article Title: Antisense tools for functional studies of human Argonaute proteins

doi: 10.1261/rna.2204610

Figure Lengend Snippet: The selected asONs show a strong inhibitory effect on hAgo mRNA expression. Dose-response curves of asON1, which is specific for hAgo1 mRNA (A), asON2, which is directed against hAgo2 mRNA (B), asON3, which was designed for hAgo3 mRNA (C), and asON4, which was selected for hAgo4 mRNA (D). The IC50 determination was performed transfecting ECV304 cells with increasing amounts of asON (0–300 nM). Cells were collected 24 h after transfection and the total RNA was isolated and reverse transcribed. The cDNA was quantified by RT–qPCR and a housekeeping gene, β glucuronidase, was used as internal control. Indicated are the hAgo mRNA levels and the IC50 values, which were calculated by using GraFit5 program. As an additional control, cells were transfected with an asON (asON_Ctr) that has no endogenous target, and the hAgo mRNA level was set at 100%. Indicated are mean values and standard deviations.

Article Snippet: The rat-anti hAgo1 used in this study, which was kindly provided by G. Meister ( Rüdel et al. 2008 ), and rat-anti hAgo2 (Ascenion) were diluted 1:10 [v/v].

Techniques: Expressing, Transfection, Isolation, Reverse Transcription, Quantitative RT-PCR, Control

Journal: RNA

Article Title: Antisense tools for functional studies of human Argonaute proteins

doi: 10.1261/rna.2204610

Figure Lengend Snippet: Down-regulation of hAgos at the protein level by specific asONs or siAgos. ECV304 cells were transfected with 100 nM asON or siAgo (specific for hAgo1, [left] or for hAgo2, [right]). As controls, 100 nM asON_Ctr or siscr were used. After 48 h, the cells were collected, lysed, and a Western blot analysis was performed using rat-anti human Ago1 and Ago2 antibodies. The hAgo proteins were seen as bands with a molecular weight of ∼100 kDa. As loading control, β actin was detected, which is shown at the bottom. The ratio between hAgo protein and β actin was normalized to asON_Ctr or siscr, and the resulting values are indicated at the bottom of the blots.

Article Snippet: The rat-anti hAgo1 used in this study, which was kindly provided by G. Meister ( Rüdel et al. 2008 ), and rat-anti hAgo2 (Ascenion) were diluted 1:10 [v/v].

Techniques: Transfection, Western Blot, Molecular Weight, Control