Journal: Oncology Letters

Article Title: Agrin is a novel oncogenic protein in thyroid cancer

doi: 10.3892/ol.2023.14070

Figure Lengend Snippet: AGRN is overexpressed in TC. (A) AGRN expression in TC (n=505) vs. normal thyroid tissue (n=59). (B) AGRN expression in THCA subtypes. The plot shows analysis of the TCGA THCA cohort data performed using UALCAN. **P<0.005, ****P<0.0001 vs. normal. (C) Expression of AGRN is associated with survival of patients with TC. The analysis was performed using UALCAN. (D) Reverse transcription-quantitative PCR analysis of AGRN mRNA expression in cell lines derived from NTHY and TC (CGTH, FTC-133 and BcPAP). Expression was analyzed in RNA isolated from one cell culture plate/cell line and three technical replicates ****P<0.0001. (E) Western blotting of AGRN protein expression in cell line derived from NTHY and TC (CGTH, FTC-133, BcPAP). Western blotting was performed on three biological replicates using monoclonal mouse anti-human AGRN antibody. The plot shows changes in AGRN protein expression normalized to β-actin. (F) Representative images of AGRN immunostaining in cell lines derived from TC (AGRN, red; phalloidin-FITC, green and DAPI, blue). Scale bar, 20 µm. TC, thyroid cancer; TCGA, The Cancer Genome Atlas; AGRN, agrin; THCA, thyroid carcinoma; UALCAN, University of Alabama at Birmingham Cancer Data Analysis Portal; PTC, papillary thyroid carcinoma; FTC, follicular thyroid carcinoma.

Article Snippet: Following washing and blocking with 2% BSA (cat. no. A9418; Sigma-Aldrich; Merk KGaA), in TBS + 0.1% Tween-20 at room temperature for 1 h, the cells were incubated with monoclonal mouse anti-human AGRN antibody (1:150; cat. no. sc-374117; Santa Cruz Biotechnology, Inc.) at 4°C overnight.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Derivative Assay, Isolation, Cell Culture, Western Blot, Immunostaining

Journal: Oncology Letters

Article Title: Agrin is a novel oncogenic protein in thyroid cancer

doi: 10.3892/ol.2023.14070

Figure Lengend Snippet: AGRN silencing suppresses migration, invasion, viability and proliferation of TC cells. (A) mRNA and (B) protein expression of AGRN in NTHY and BcPAP cells transfected with siAGRN or NEG were determined using reverse transcription-quantitative PCR and western blotting, respectively. *P<0.05, **P<0.0016 and ***P<0.0006. The plot shows changes in protein expression that were normalized to β-actin. The effects of siAGRN on (C) migration and (D) invasion of NTHY and BcPAP cells. The plots show modified Boyden chamber migration and invasion assays performed in three independent biological experiments. Magnification, ×200. (E) Effects of siAGRN transfection on vimentin, N-cadherin and E-cadherin protein levels in NTHY and BcPAP cells. *P<0.05 and **P<0.005. Representative western blotting of protein expression normalized GAPDH. (F) Effects of siAGRN transfection on the viability of NTHY and BcPAP cells. The plots show the results of the MTS assay performed in three independent biological experiments. *P<0.05. (G) Effects of siAGRN on the proliferation of NTHY and BcPAP cells. The plots show BrdU assay performed in three independent biological experiments. ***P<0.0002 and *P<0.01. TC, thyroid cancer; siRNA, small interfering RNA; NEG, negative control; AGRN, agrin.

Article Snippet: Following washing and blocking with 2% BSA (cat. no. A9418; Sigma-Aldrich; Merk KGaA), in TBS + 0.1% Tween-20 at room temperature for 1 h, the cells were incubated with monoclonal mouse anti-human AGRN antibody (1:150; cat. no. sc-374117; Santa Cruz Biotechnology, Inc.) at 4°C overnight.

Techniques: Migration, Expressing, Transfection, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Modification, MTS Assay, BrdU Staining, Small Interfering RNA, Negative Control

Journal: Oncology Letters

Article Title: Agrin is a novel oncogenic protein in thyroid cancer

doi: 10.3892/ol.2023.14070

Figure Lengend Snippet: Expression of AGRN is associated with neutrophil infiltration in THCA. (A) Analysis performed using Timer. (B) Lack of a correlation between the expression of AGRN and T cells or inflammatory cells in THCA (n=509). AGRN, agrin; THCA, thyroid carcinoma; TPM, transcripts per million.

Article Snippet: Following washing and blocking with 2% BSA (cat. no. A9418; Sigma-Aldrich; Merk KGaA), in TBS + 0.1% Tween-20 at room temperature for 1 h, the cells were incubated with monoclonal mouse anti-human AGRN antibody (1:150; cat. no. sc-374117; Santa Cruz Biotechnology, Inc.) at 4°C overnight.

Techniques: Expressing

Journal: PLoS ONE

Article Title: C. elegans Agrin Is Expressed in Pharynx, IL1 Neurons and Distal Tip Cells and Does Not Genetically Interact with Genes Involved in Synaptogenesis or Muscle Function

doi: 10.1371/journal.pone.0000731

Figure Lengend Snippet: A, Schematic representation of the recombinant fragments used as antigen to raise monoclonal and polyclonal antibodies. For eukaryotic expression the C-terminal lamG domains were fused to a short fragment of chicken tenascin C (Tn-C), including a secretion signal and the epitope of the anti-Tn60 antibody. The specificity of both polyclonal and monoclonal antibodies was tested by western blotting on conditioned medium of COS cells transfected with the construct encoding the two LamG domains with the Tn-C-tag (B). Lanes 1 and 2 were incubated with polyclonal antisera from two different rabbits, lane 3 with the monoclonal antibody pool raised against the bacterially expressed fragment, and lane 4 with pre-immune serum. All antibodies detected a band of about 80 kDa, which corresponds to the size of the recombinant protein. Additional smaller bands (asterisk) most likely correspond to degradation products which are not recognized by the monoclonal antibodies. C–J, Immunofluorescence staining of transfected COS cells was performed with the anti-agrin monoclonal antibody pool (C–F) and compared to the anti-Tn60 control (G–J). In transfected cells (C and D; G and H) the secreted agrin fragment was detected on cell surfaces of non-permeabilized cells (C and G) or in the endoplasmatic reticulum/Golgi apparatus of permeabilized cells (D and H). Non-transfected cells were used as a negative control (E and F; I and J).

Article Snippet: For agrin monoclonal antibodies anti-mouse secondary antibody conjugated with a green dye (Alexa Fluor ® 488, Molecular Probes), for polyclonal anti-agrin secondary anti-rabbit labeled green (Alexa Fluor ® 488, Molecular Probes) and for rim polyclonal antibody red-labeled anti-rabbit (Alexa Fluor ® 546, Molecular Probes) was used.

Techniques: Recombinant, Expressing, Western Blot, Transfection, Construct, Incubation, Immunofluorescence, Staining, Negative Control