Journal: Cell Death & Disease

Article Title: β1,4-galactosyltransferase III drives retinoblastoma invasion via activation of integrin-FAK axis

doi: 10.1038/s41419-026-08620-5

Figure Lengend Snippet: A Metabolic pathway activities across cell types in integrated retinoblastoma (RB) datasets (4 RB samples from GSE249995 and 7 from GSE168434 ). Pathways with non-significant activity (permutation test, P > 0.05) are shown as blank. B AB-PAS staining of a human RB paraffin section (International Intraocular Retinoblastoma Classification [IIRC] stage E). C , D . Dot plots showing normalized expression of genes involved in the glycosaminoglycan biosynthesis–keratan sulfate pathway in different cell types from extraocular ( C ) and intraocular ( D ) RB samples. E UMAP visualization of B4GALT1 − 4 expression in RB cells. F Comparison of B4GALT family gene expression across retina, intraocular RB, and extraocular RB based on scRNA-seq data. G , H qPCR ( G ) and western blot ( H ) analyses demonstrating significantly elevated B4GALT3 mRNA and protein levels in RB cell lines (WERI-Rb1 and Y79) compared to human retinal pigment epithelial cells (ARPE-19). I Immunofluorescence co-staining of B4GALT3 and Ki67 in proliferative tumor regions of IIRC stage E RB and orthotopic xenografts. Data are presented as mean ± SD from 3 independent experiments. Statistical significance in ( G ) was determined by one-way ANOVA. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: The cell lines utilized in this research included retinoblastoma cell lines WERI-Rb1 (HTB-169, American Type Culture Collection [ATCC]) and Y79 (HTB-18, ATCC), human embryonic kidney cell line HEK293T (CRL-3216, ATCC), and human retinal pigment epithelial cell line ARPE-19 (CRL-2302, ATCC).

Techniques: Activity Assay, Staining, Paraffin Section, Expressing, Comparison, Gene Expression, Western Blot, Immunofluorescence

Journal: Cell Death & Disease

Article Title: β1,4-galactosyltransferase III drives retinoblastoma invasion via activation of integrin-FAK axis

doi: 10.1038/s41419-026-08620-5

Figure Lengend Snippet: A Western blot analysis showing decreased B4GALT3 protein levels in WERI-Rb1 and Y79 cells upon knockdown using two independent shRNAs. B , C CCK-8 proliferation assays in RB cell lines WERI-Rb1 ( B ) and Y79 ( C ) following B4GALT3 knockdown with shRNA for 24–72 h. D Representative images and quantification of EdU incorporation assay in RB cell lines after B4GALT3 knockdown for 48 h. E Volcano plot depicting differentially expressed genes (DEGs) from RNA-seq analysis comparing WERI-Rb1 cells treated with control shRNA (shNC) and shB4GALT3. F Top 10 enriched pathways based on KEGG analysis of downregulated DEGs in WERI-Rb1 cells following B4GALT3 knockdown. G Representative immunofluorescence (IF) staining images demonstrating co-localization of β1-integrin and B4GALT3 in orthotopic xenograft sections. H , I Analysis of B4GALT3-modified glycosylation of β1-integrin in RB cells. Cell lysates from WERI-Rb1 (H) and Y79 (I) cells were subjected to RCA I pull-down (PD) followed by Western blot with an anti-β1-integrin antibody. J , K Western blot analysis showing alterations in the FAK-PI3K-AKT signaling pathway in WERI-Rb1 ( J ) and Y79 ( K ) cells following B4GALT3 knockdown. L Representative images and quantification of fibronectin adhesion assay in RB cells following B4GALT3 knockdown. Data are presented as mean ± SD from three independent experiments. Statistical significance in ( B , C , D , L ) was determined by one-way ANOVA. ns , no statistical difference; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: The cell lines utilized in this research included retinoblastoma cell lines WERI-Rb1 (HTB-169, American Type Culture Collection [ATCC]) and Y79 (HTB-18, ATCC), human embryonic kidney cell line HEK293T (CRL-3216, ATCC), and human retinal pigment epithelial cell line ARPE-19 (CRL-2302, ATCC).

Techniques: Western Blot, Knockdown, CCK-8 Assay, shRNA, RNA Sequencing, Control, Immunofluorescence, Staining, Modification, Glycoproteomics, Cell Adhesion Assay

Journal: Cell Death & Disease

Article Title: β1,4-galactosyltransferase III drives retinoblastoma invasion via activation of integrin-FAK axis

doi: 10.1038/s41419-026-08620-5

Figure Lengend Snippet: A Western blot analysis showing increased B4GALT3 protein levels in WERI-Rb1 cells following B4GALT3 overexpression. B CCK-8 proliferation assays in WERI-Rb1 cells following B4GALT3 overexpression for 24–72 h. C Representative images and quantification of EdU incorporation assay assessing proliferation in WERI-Rb1 cells after B4GALT3 overexpression for 48 h. D Representative images and quantification of fibronectin adhesion assay in WERI-Rb1 cells following B4GALT3 overexpression. E Analysis of B4GALT3-overexpression-induced glycosylation of β1-integrin in WERI-Rb1 cells. F Western blot analysis showing alterations in the FAK-PI3K-AKT signaling pathway in WERI-Rb1 cells following B4GALT3 overexpression. G Western blot analysis showing alterations of β1-integrin in B4GALT3-overexpressing WERI-Rb1 cells treated with FAK inhibition. H – J Western blot analysis showing alterations of the FAK-PI3K-AKT signaling pathway in B4GALT3-overexpressing WERI-Rb1 cells treated with FAK inhibition. Quantification of total FAK (tFAK) and phosphorylated FAK (pFAK) ( H ), total PI3K (tPI3K) and phosphorylated PI3K (pPI3K) ( I ), and total AKT (tAKT) and phosphorylated AKT (pAKT) ( J ). K , L Representative images ( K ) and quantification ( L ) of fibronectin adhesion assay in B4GALT3-overexpressing WERI-Rb1 cells treated with FAK inhibitor. Data are presented as mean ± SD from three independent experiments. Statistical significance in ( B , C , D , L ) was determined by a two-tailed unpaired t -test. ns , no statistical difference; * P < 0.05; ** P < 0.01; **** P < 0.0001.

Article Snippet: The cell lines utilized in this research included retinoblastoma cell lines WERI-Rb1 (HTB-169, American Type Culture Collection [ATCC]) and Y79 (HTB-18, ATCC), human embryonic kidney cell line HEK293T (CRL-3216, ATCC), and human retinal pigment epithelial cell line ARPE-19 (CRL-2302, ATCC).

Techniques: Western Blot, Over Expression, CCK-8 Assay, Cell Adhesion Assay, Glycoproteomics, Inhibition, Two Tailed Test

Journal: Cell Death & Disease

Article Title: β1,4-galactosyltransferase III drives retinoblastoma invasion via activation of integrin-FAK axis

doi: 10.1038/s41419-026-08620-5

Figure Lengend Snippet: A Representative immunofluorescence (IF) staining image demonstrating co-localization of MMP2 and B4GALT3 in human IIRC stage E RB sections. B Western blot analysis of MMP2 expression in WERI-Rb1 cells following B4GALT3 knockdown or overexpression. C MMP2 mRNA expression in RNA-seq data of WERI-Rb1 cells treated with control shRNA (shNC) or shB4GALT3. D Gelatin zymography assay showing the levels of active MMP2 in the supernatants of WERI-Rb1 cells with B4GALT3 knockdown or overexpression. E Schematic diagram of a co-culture system of RB cells and ARPE-19 retinal epithelial cells to model tumor invasion across the outer blood–retinal barrier. F Western blot analysis of ZO-1 and occludin in ARPE-19 cells co-cultured with WERI-Rb1 cells under B4GALT3 modulation (knockdown or overexpression). G Representative immunofluorescence images and quantification of ZO-1 and occludin in ARPE-19 co-cultures with B4GALT3-modulated WERI-Rb1 cells. H Western blot analysis of MMP2 expression in B4GALT3-overexpressing WERI-Rb1 cells treated with a FAK inhibitor. I Representative images and quantification of ZO-1 and occludin immunofluorescence staining in ARPE-19 cells co-cultured with B4GALT3-overexpressing WERI-Rb1 cells, following FAK and MMP inhibition. J Western blot analysis of ZO-1 and occludin in ARPE-19 co-cultures under the same conditions as in ( I ). Data are presented as mean ± SD from three independent experiments. Statistical significance was determined by a two-tailed unpaired t -test for ( C ), and one-way ANOVA for ( G ) and ( I ). ns , no statistical difference; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: The cell lines utilized in this research included retinoblastoma cell lines WERI-Rb1 (HTB-169, American Type Culture Collection [ATCC]) and Y79 (HTB-18, ATCC), human embryonic kidney cell line HEK293T (CRL-3216, ATCC), and human retinal pigment epithelial cell line ARPE-19 (CRL-2302, ATCC).

Techniques: Immunofluorescence, Staining, Western Blot, Expressing, Knockdown, Over Expression, RNA Sequencing, Control, shRNA, Zymography Assay, Co-Culture Assay, Cell Culture, Inhibition, Two Tailed Test

Journal: Cell Death & Disease

Article Title: β1,4-galactosyltransferase III drives retinoblastoma invasion via activation of integrin-FAK axis

doi: 10.1038/s41419-026-08620-5

Figure Lengend Snippet: A Schematic workflow of high-throughput virtual screening (HTVS) for identifying potential B4GALT3 inhibitors. B Docking scores of the top 15 candidate compounds identified from the HTVS. C Quantification of cell viability in RB cell lines (WERI-Rb1 and Y79) treated with the top 15 candidate compounds (100 μM) for 24 h. D , E In silico docking of Myricoside into the active site of human B4GALT3 protein ( D ), highlighting the detailed molecular interactions within the binding pocket ( E ). F Cellular thermal shift assay (CETSA) curves showing thermal stabilization of B4GALT3 protein in RB cell lysates with or without myricoside treatment (100 μM). G , H Representative images and quantification of ( G ) propidium iodide (PI)-positive cells and (H) fibronectin-adherent cells in RB cell lines treated with increasing concentrations of myricoside for 48 h. I Western blot analysis showing alterations in β1-integrin glycosylation in RB cells treated with myricoside (100 μM). J Western blot analysis of the FAK–PI3K–AKT signaling pathway in RB cells following myricoside treatment (100 μM). Data are presented as mean ± SD from three independent experiments. Statistical significance was determined by one-way ANOVA for ( G ) and ( H ). Exact P -values are indicated in the corresponding figures.

Article Snippet: The cell lines utilized in this research included retinoblastoma cell lines WERI-Rb1 (HTB-169, American Type Culture Collection [ATCC]) and Y79 (HTB-18, ATCC), human embryonic kidney cell line HEK293T (CRL-3216, ATCC), and human retinal pigment epithelial cell line ARPE-19 (CRL-2302, ATCC).

Techniques: High Throughput Screening Assay, In Silico, Binding Assay, Thermal Shift Assay, Western Blot, Glycoproteomics