Journal: Frontiers in Endocrinology

Article Title: Novel Compound Heterozygous Pathogenic Mutations of SLC5A5 in a Chinese Patient With Congenital Hypothyroidism

doi: 10.3389/fendo.2021.620117

Figure Lengend Snippet: Characterization of cellular localization of G421R and G51fs NIS mutants in 293T cells. pEGFP-N2 empty vector was employed as a negative control (Mock). Human WT NIS, D331N, G421R, and G51fs mutants were cloned into pEGFP-N2 plasmids (green). The cell membrane was labeled with a fluorescent probe Dil (red). Mock, WT NIS, D331N, G421R, and G51fs plasmids were transfected into 293T cells 24 h after seeding. Mutant D331N NIS showed slightly decreased membrane fluorescence, compared with the WT. However, G51fs and G421R mutants displayed severely reduced cell membrane localization in 293T cells. Nuclei were stained with DAPI (blue). Scale: 10 μm.

Article Snippet: Similarly, a WT NIS-EGFP fusion protein was expressed in a pEGFP-N2 expression vector (TransGen Biotech) using EcoRI and BamHI restriction sites, and the GFP was tagged downstream to the NIS coding region.

Techniques: Plasmid Preparation, Negative Control, Clone Assay, Labeling, Transfection, Mutagenesis, Fluorescence, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: EDF1 accelerates ganglioside GD3 accumulation to boost CD52-mediated CD8 + T cell dysfunction in neuroblastoma

doi: 10.1186/s13046-025-03307-9

Figure Lengend Snippet: EDF1 accelerates GD3 secretion of NB cells and inhibit the functions of CD8 + T cells by recruiting to mitochondrial microdomain. A Correlation plots among EDF1 and synthases for gangliosides in an RNA sequencing matrix involving Ctrl, EDF1-overexpressing and EDF1-knockdown SK-N-AS cells. B qRT‒PCR analyses of synthases for gangliosides in EDF1-altered NB cells. C Immunoblotting analyses of ST8SIA1 and ST3GAL5 in EDF1-altered NB cells. β-Actin was used as a loading control for immunoblotting. Ctrl in A-C stands for the average of undistinguishable controls of scrambled sequence (for Sh-EDF1) and empty vector (for EDF1 overexpression). D Representative immunofluorescence staining images of ST8SIA1 (green) and GD3 (red) in EDF1-altered NB cells. DAPI (blue) was used to stain nuclei. E The chemotaxis of CD8 + T cells induced by EDF1-altered NB cells was analyzed by counting migrated cells in the bottom chamber of transwell inserts. F , G Representative flow cytometry histograms of CD52 ( F ) and ROS levels ( G ), in CD8 + T cells exposed to exogenous GD3. The percentages of positive staining were statistically analyzed. ( H-J ) Representative flow cytometry plots of apoptosis ( H ) and mRNA levels of apoptosis-related genes in CD8 + T cells exposed to exogenous GD3. The percentages of living cells were statistically analyzed in ( I ). ( K ) Representative flow cytometry histograms of GD3 in CD8 + T cells with or without permeabilization exposing to exogenous GD3. The percentages of positive staining were statistically analyzed. ( L ) Representative immunofluorescence staining images of COX2 (green, mitochondrial inner membrane) and GD3 (red) treated as indicated. DAPI (blue) was used to stain nuclei. The scale bar represents 20 µm. The data in B-L represent the mean ± SD ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Small interfering RNAs (siRNA) targeting EDF1 and ST8SIA1 were separately used to transiently knockdown the indicated genes (RiboBio Co., Ltd, Guangzhou, China), and a control transfected with scrambled sequence was used in the study.

Techniques: RNA Sequencing, Knockdown, Western Blot, Control, Sequencing, Plasmid Preparation, Over Expression, Immunofluorescence, Staining, Chemotaxis Assay, Flow Cytometry, Membrane

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: EDF1 accelerates ganglioside GD3 accumulation to boost CD52-mediated CD8 + T cell dysfunction in neuroblastoma

doi: 10.1186/s13046-025-03307-9

Figure Lengend Snippet: EDF1 accelerates NF-κB/RelA binding to the promoter of ST8SIA1 to avoid methylation. A Representative immunofluorescence staining images of ST8SIA1 (green) and GD3 (red) treated as indicated. DAPI (blue) was used to stain nuclei. B , E , F , H Immunoblot analysis of ST8SIA1 expression in cells treated as indicated. β-Actin was used as a loading control. C , D Coimmunoprecipitation (Co-IP) assays were performed to verify the interaction between NF-κB/RelA and EDF1. G ChIP-PCR assays showed that 54 ~ −63 bp of the ST8SIA1 promoter contains a consensus NF-κB/RelA binding region. I , J Immunoblot analysis of NF-κB/RelA, ST8SIA1 and EDF1 expression in the cytoplasm and nucleus of NB cells, as well as their total levels after treatment with PMA. β-Actin was used as a loading control in both the cell cytoplasm and total cell lysates, and histone H3 was used as a loading control separately in the cell nucleus. The data in G represent the mean ± SD ( n = 3). *** P < 0.001

Article Snippet: Small interfering RNAs (siRNA) targeting EDF1 and ST8SIA1 were separately used to transiently knockdown the indicated genes (RiboBio Co., Ltd, Guangzhou, China), and a control transfected with scrambled sequence was used in the study.

Techniques: Binding Assay, Methylation, Immunofluorescence, Staining, Western Blot, Expressing, Control, Co-Immunoprecipitation Assay