Journal: NPJ Breast Cancer

Article Title: Targeting GD2 with naxitamab overcomes GD3 synthase-driven immune suppression in triple-negative breast cancer

doi: 10.1038/s41523-025-00843-7

Figure Lengend Snippet: A Analysis of GD3S ( ST8SIA1 ) mRNA expression in METABRIC and TCGA (pan cancer) BC patient datasets shows significant correlation with immune checkpoint inhibitors. In the METABRIC dataset, Pearson’s correlation coefficients indicate significant correlations of GD3S mRNA expression with CTLA-4 ( r = 0.4; P < 0.001), LAG3 ( r = 0.33; P < 0.001), PD1 ( r = 0.3; P < 0.001), CD25 ( r = 0.38; P < 0.001), and SIRPα ( r = 0.36; P < 0.001). Similarly, in the TCGA dataset, GD3S mRNA expression was significantly correlated with CTLA-4 ( r = 0.47; P < 0.001), LAG3 ( r = 0.33; P < 0.001), PD1 ( r = 0.38; P < 0.001), CD25 ( r = 0.45; P < 0.001), and SIRPα ( r = 0.56; P < 0.001). B Representative IHC images obtained by sequential multiplex immunofluorescence images (Lunaphore COMET) from FFPE samples of TNBC patients with high or low GD3S expression ( n = 20 total) were used to compare immune infiltration patterns, showing distinct fluorescence intensities for PANK, Ki67, CD163, CD86, CD56, CD8, CD4, FOXP3, and GD3S within tumor tissues. C H-scores for GD3S expression in BC patients categorized into high and low GD3S expression groups ( ****P < 0.0001). Immune cell counts using Visiopharm IHC image analysis software show differences in the expression of CD8, CD4, CD86, and CD56 between the high and low GD3S expression groups ( ****P < 0.0001; **P < 0.01). Scale bar in IHC images represents 55 µm with 20x magnification. Bar graphs display means ± standard deviations.

Article Snippet: We also purchased the following antibodies: pan-Cytokeratin (Proteintech, cat. #26411-1-AP, rabbit host) along with GD3S (Proteintech, cat. #24918-1-AP, rabbit host), CD45 (Fortislife, clone BL-178-1267, cat. #A700-012, rabbit host), Ki67 (Bethyl, clone BLR021E, cat. #A700-021CF, rabbit host), CD3 (Fortislife, clone BL-298-5012, cat. #A700-016F, rabbit host), CD8 (Fortislife, clone c8/144B, cat. #A500-012A, mouse host), CD4 (Bethyl, clone BL-155-1C11, cat. #A700-015, rabbit host), FOXP3 (Bethyl, clone BLR034F, cat. #A700-034, rabbit host), CD68 (Fortislife, cat. #A500-018ACF, mouse host), CD163 (Invitrogen, clone I0D6, cat. #MA5-11458, mouse host), CD86 (CST, clone E2G8P, cat. #76755SF, rabbit host), CD56 (Fortislife, clone BLR152J, cat. #A700-152, rabbit host) and human IgG1 Kappa antibody (hIgG, cat. #0151K-14, SouthernBiotech).

Techniques: Expressing, Multiplex Assay, Immunofluorescence, Fluorescence, Software

Journal: NPJ Breast Cancer

Article Title: Targeting GD2 with naxitamab overcomes GD3 synthase-driven immune suppression in triple-negative breast cancer

doi: 10.1038/s41523-025-00843-7

Figure Lengend Snippet: A , B MCF7, BT549, and SUM159 cells were genetically modified to stably overexpress GD3 synthase or an empty vector (EV) control plasmid. These stable cell lines were co-cultured with macrophages (Φ) derived from healthy donors in 1:1 E:T ratios, and macrophage-mediated phagocytosis was measured on the basis of changes in red fluorescence intensity using the pH-sensitive pHrodo dye. A Bar graph shows the analysis of GD3S overexpression on pHrodo fluorescence intensity at the final timepoint (shown in Fig. ), reflecting the level of macrophage-mediated phagocytosis ( ***P < 0.001; **P < 0.01). B Representative IncuCyte images display the differences in fluorescence between GD3S-overexpressing cells and EV controls co-cultured with macrophages. C , D GD3S-overexpressing MCF7, BT549, and SUM159 cells and their respective control cells were co-cultured with NK cells derived from healthy donors in 8:1 E:T ratios, and NK cell–mediated apoptosis was measured by changes in green fluorescence intensity using fluorescently tagged annexin V. C Bar graph shows the effect of GD3S overexpression on NK cell–mediated target cell apoptosis at the final timepoint shown in Fig. ( ****P < 0.0001; ***P < 0.001). D Representative IncuCyte images show differences in fluorescence intensity due to annexin V binding in GD3S-overexpressing cells and EV controls co-cultured with NK cells. E , F GD3S-overexpressing MCF7, BT549, and SUM159 cells and their respective control cells were co-cultured with T cells derived from healthy donors in 8:1 E:T ratios, and T cell–mediated cytotoxicity was measured by changes in green fluorescence intensity using the cytotox green dye. E Bar graph shows the effect of GD3S overexpression on T cell–mediated target cell cytotoxicity at the final timepoint shown in Fig. ( **P < 0.01; *P < 0.05). F Representative IncuCyte images show differences in fluorescence intensity due to cytotox green levels in GD3S-overexpressing cells and EV controls co-cultured with T cells. Scale bar in IncuCyte images represents 400 µm.

Article Snippet: We also purchased the following antibodies: pan-Cytokeratin (Proteintech, cat. #26411-1-AP, rabbit host) along with GD3S (Proteintech, cat. #24918-1-AP, rabbit host), CD45 (Fortislife, clone BL-178-1267, cat. #A700-012, rabbit host), Ki67 (Bethyl, clone BLR021E, cat. #A700-021CF, rabbit host), CD3 (Fortislife, clone BL-298-5012, cat. #A700-016F, rabbit host), CD8 (Fortislife, clone c8/144B, cat. #A500-012A, mouse host), CD4 (Bethyl, clone BL-155-1C11, cat. #A700-015, rabbit host), FOXP3 (Bethyl, clone BLR034F, cat. #A700-034, rabbit host), CD68 (Fortislife, cat. #A500-018ACF, mouse host), CD163 (Invitrogen, clone I0D6, cat. #MA5-11458, mouse host), CD86 (CST, clone E2G8P, cat. #76755SF, rabbit host), CD56 (Fortislife, clone BLR152J, cat. #A700-152, rabbit host) and human IgG1 Kappa antibody (hIgG, cat. #0151K-14, SouthernBiotech).

Techniques: Genetically Modified, Stable Transfection, Plasmid Preparation, Control, Cell Culture, Derivative Assay, Fluorescence, Over Expression, Binding Assay

Journal: NPJ Breast Cancer

Article Title: Targeting GD2 with naxitamab overcomes GD3 synthase-driven immune suppression in triple-negative breast cancer

doi: 10.1038/s41523-025-00843-7

Figure Lengend Snippet: A SUM159 and BT549 cells were genetically modified to knockout GD3S ( ST8SIA1 ) using the CRISPR-Cas9 system. These GD3S-KO cells and their respective control cells were co-cultured with macrophages (Φ) derived from healthy donors in 1:1 E:T ratios, and macrophage-mediated phagocytosis was measured on the basis of changes in red fluorescence intensity using the pH-sensitive pHrodo dye. Bar graph shows the effect of GD3S-KO on pHrodo fluorescence intensity at the final timepoint (shown in Fig. ), reflecting the level of macrophage-mediated phagocytosis ( **P < 0.01; ***P < 0.001). Representative IncuCyte images display the differences in fluorescence between GD3S-KO and control cells, co-cultured with macrophages. B GD3S-KO SUM159 and BT549 cells and their respective control cells were co-cultured with NK cells derived from healthy donors in 8:1 E:T ratios, and NK cell–mediated apoptosis was measured by changes in green fluorescence intensity using fluorescently tagged annexin V. Bar graph shows the effect of GD3S-KO on NK cell–mediated target cell apoptosis at the final timepoint shown in Fig. ( ****P < 0.0001). Representative IncuCyte images show differences in fluorescence intensity due to annexin V binding in GD3S-KO and control cells, co-cultured with NK cells. C GD3S-KO SUM159 and BT549 cells and their respective control cells were co-cultured with T cells derived from healthy donors in 8:1 E:T ratios, and T cell–mediated cytotoxicity was measured by changes in green fluorescence intensity using the cytotox green dye. Bar graph shows the effect of GD3S-KO on T cell–mediated target cell cytotoxicity at the final timepoint shown in Fig. ( ****P < 0.0001). Representative IncuCyte images show differences in fluorescence intensity due to cytotox green levels in GD3S-KO and control cells, co-cultured with T cells. Scale bar in IncuCyte images represents 400 µm.

Article Snippet: We also purchased the following antibodies: pan-Cytokeratin (Proteintech, cat. #26411-1-AP, rabbit host) along with GD3S (Proteintech, cat. #24918-1-AP, rabbit host), CD45 (Fortislife, clone BL-178-1267, cat. #A700-012, rabbit host), Ki67 (Bethyl, clone BLR021E, cat. #A700-021CF, rabbit host), CD3 (Fortislife, clone BL-298-5012, cat. #A700-016F, rabbit host), CD8 (Fortislife, clone c8/144B, cat. #A500-012A, mouse host), CD4 (Bethyl, clone BL-155-1C11, cat. #A700-015, rabbit host), FOXP3 (Bethyl, clone BLR034F, cat. #A700-034, rabbit host), CD68 (Fortislife, cat. #A500-018ACF, mouse host), CD163 (Invitrogen, clone I0D6, cat. #MA5-11458, mouse host), CD86 (CST, clone E2G8P, cat. #76755SF, rabbit host), CD56 (Fortislife, clone BLR152J, cat. #A700-152, rabbit host) and human IgG1 Kappa antibody (hIgG, cat. #0151K-14, SouthernBiotech).

Techniques: Genetically Modified, Knock-Out, CRISPR, Control, Cell Culture, Derivative Assay, Fluorescence, Binding Assay

Journal: NPJ Breast Cancer

Article Title: Targeting GD2 with naxitamab overcomes GD3 synthase-driven immune suppression in triple-negative breast cancer

doi: 10.1038/s41523-025-00843-7

Figure Lengend Snippet: A Schematic of the ganglioside biosynthetic pathway, illustrating the synthesis of 0-, A-, B-, and C-series gangliosides starting from the ceramide backbone, with sequential enzymatic additions of glucose, galactose, N-acetyl-D-galactosamine, and N-acetylneuraminic acid moieties. The highlighted section shows GD3 synthase ( ST8SIA1 ) converting GM3 to GD3, leading to downstream B- and C-series gangliosides. B Schematic representation of lentivirus-mediated generation of BC cell lines stably overexpressing GD3 synthase (pLenti-III-ST8SIA-CMV plasmid). C Heat map displaying results of mass spectrometry–based lipidomic analysis in GD3S-overexpressing (GD3S-OE) SUM159 and MCF7 cells compared to in their EV-control cells. GD3S-OE cells show increased levels of gangliosides GD2 and GD3 (in red) and decreased levels of GM3 (in blue). Each row represents a metabolite within the lipid class, and columns correspond to samples. D Volcano plot illustrating metabolite enrichment, with individual metabolites represented by their p value and log2 fold change between GD3S-OE and EV-control cells. Color intensity of the dots reflects metabolite levels determined by mass spectrometry imaging (MSI) annotation. GD2 and GD3 (highlighted in blue) are highly enriched compared to GM2 and GM3. E mRNA quantification by quantitative polymerase chain reaction shows significantly elevated GD3S expression in GD3S-OE SUM159, BT549, and MCF7 cells ( ****P < 0.0001; **P < 0.01). F A flow cytometry analysis indicates that GD3S-OE cells have over 90% GD2 expression compared to EV-control cells, which show GD2 levels of 9.99%, 11.1%, and 2.28% in SUM159, BT549, and MCF7 cells, respectively.

Article Snippet: We also purchased the following antibodies: pan-Cytokeratin (Proteintech, cat. #26411-1-AP, rabbit host) along with GD3S (Proteintech, cat. #24918-1-AP, rabbit host), CD45 (Fortislife, clone BL-178-1267, cat. #A700-012, rabbit host), Ki67 (Bethyl, clone BLR021E, cat. #A700-021CF, rabbit host), CD3 (Fortislife, clone BL-298-5012, cat. #A700-016F, rabbit host), CD8 (Fortislife, clone c8/144B, cat. #A500-012A, mouse host), CD4 (Bethyl, clone BL-155-1C11, cat. #A700-015, rabbit host), FOXP3 (Bethyl, clone BLR034F, cat. #A700-034, rabbit host), CD68 (Fortislife, cat. #A500-018ACF, mouse host), CD163 (Invitrogen, clone I0D6, cat. #MA5-11458, mouse host), CD86 (CST, clone E2G8P, cat. #76755SF, rabbit host), CD56 (Fortislife, clone BLR152J, cat. #A700-152, rabbit host) and human IgG1 Kappa antibody (hIgG, cat. #0151K-14, SouthernBiotech).

Techniques: Stable Transfection, Plasmid Preparation, Mass Spectrometry, Control, Imaging, Real-time Polymerase Chain Reaction, Expressing, Flow Cytometry

Journal: Journal of Virology

Article Title: β-Catenin, a Transcription Factor Activated by Canonical Wnt Signaling, Is Expressed in Sensory Neurons of Calves Latently Infected with Bovine Herpesvirus 1

doi: 10.1128/JVI.02971-15

Figure Lengend Snippet: ORF2 and β-catenin are frequently expressed in the same neuron during latency. Consecutive sections were cut from formalin-fixed paraffin-embedded TG from latently infected calves. (A) For one section, IHC was performed using a peptide-specific ORF2 antibody (1:250 dilution) that has been previously described (16, 22, 42, 43). The adjacent section was stained with the β-catenin antibody (1:200 dilution). Biotinylated goat anti-rabbit IgG (Vector Laboratories) was used as a secondary antibody. Neurons, indicated by numbers 1 to 9, are ORF2+ and β-catenin+ neurons. These results are representative of results from many sections using TG from two calves. (B) For one section, IHC was performed using the β-catenin antibody (1:200 dilution). The consecutive section was stained using a peptide-specific bICP0 antibody (1:250 dilution) that specifically recognizes the bICP0 protein (52, 53). Neurons, indicated by numbers 1 to 12, are β-catenin+ neurons in one section and are also shown in the section stained with the bICP0 antibody to point out the location of the same neurons numbered in the section stained by the β-catenin antibody. These results are representative of four sections cut from TG of two latently infected calves.

Article Snippet: Forty-eight hours after transfection cells were collected and processed for Western blot analysis using a Flag antibody (1:1,000 dilution; Sigma) to detect ORF2, a β-catenin antibody (1:5000 dilution), or an anti-V5 antibody (1:5,000 dilution; Rockland) to detect MAML1.

Techniques: Formalin-fixed Paraffin-Embedded, Infection, Staining, Plasmid Preparation

Journal: Journal of Virology

Article Title: β-Catenin, a Transcription Factor Activated by Canonical Wnt Signaling, Is Expressed in Sensory Neurons of Calves Latently Infected with Bovine Herpesvirus 1

doi: 10.1128/JVI.02971-15

Figure Lengend Snippet: ORF2 and MAML1 stabilized β-catenin and NICD3 protein levels in transfected Neuro-2A cells. (A) Neuro-2A cells were transfected with the designated plasmids (2 μg of DNA for each plasmid). An empty CMV expression vector (pcDNA3.1) was added to maintain equal amounts of plasmid transfected. Forty-eight hours after transfection cells were collected and processed for Western blot analysis using a Flag antibody (1:1,000 dilution; Sigma) to detect ORF2, a β-catenin antibody (1:5000 dilution), or an anti-V5 antibody (1:5,000 dilution; Rockland) to detect MAML1. Fifty micrograms of protein in the total cell lysate was used for each lane. β-Tubulin was used as a loading control. (B) Levels of β-catenin protein in the respective samples presented in panel A were estimated by comparing levels of tubulin to those of β-catenin using a Bio-Rad molecular imager. β-Catenin protein levels in samples transfected with just the β-catenin expression plasmid were normalized to 1, and other values were compared to this result. The results shown in this graph were derived from three independent studies. (C) Neuro-2A cells were transfected with 1 μg of MAML1 expression plasmid alone or with 1 μg to 3 μg of ORF2 as indicated. Forty-eight hours after transfection cells were collected and processed for Western blot analysis using a Flag antibody (1:1,000 dilution; Sigma) or V5 antibody (1:5,000 dilution; Rockland). Fifty micrograms of protein in the total cell lysate was used for each lane, and β-tubulin was used as a loading control. (D) Neuro-2A cells were transfected with DNA constructs (2 μg of DNA for each plasmid) as indicated, and the empty expression vector was added as needed to maintain equal amounts of plasmid. Forty-eight hours after transfection, cells were collected and processed for Western blot analysis using the indicated antibodies at the same concentrations as for the experiments shown in panels A and B. The anti-Notch3 antibody (1:300) (Santa Cruz) was used to detect NICD3. The results shown in panels A, C, and D are representative of four independent experiments. NT, not treated.

Article Snippet: Forty-eight hours after transfection cells were collected and processed for Western blot analysis using a Flag antibody (1:1,000 dilution; Sigma) to detect ORF2, a β-catenin antibody (1:5000 dilution), or an anti-V5 antibody (1:5,000 dilution; Rockland) to detect MAML1.

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Control, Derivative Assay, Construct

Journal: Journal of Virology

Article Title: β-Catenin, a Transcription Factor Activated by Canonical Wnt Signaling, Is Expressed in Sensory Neurons of Calves Latently Infected with Bovine Herpesvirus 1

doi: 10.1128/JVI.02971-15

Figure Lengend Snippet: ORF2 stimulates transactivation potential of MAML1 for β-catenin and NICD-dependent transcription. (A) Neuro-2A cells were cotransfected with 0.1 μg of TOPFlash luciferase reporter, 0.1 μg of β-catenin (S33Y), and 0.1 μg of ORF2. (B) Neuro-2A cells were cotransfected with 0.1 μg of TOPFlash luciferase reporter, 0.1 μg of S33Y (β-catenin), 0.1 μg of MAML1, and increasing concentrations of ORF2 (0.1 to 0.3 μg). (C and D) Neuro-2A cells were cotransfected with 0.3 μg of an HES1 luciferase reporter, 0.3 μg of NICD1 or NICD3, different concentrations of the MAML1 plasmid (0.1 μg and 0.3 μg), and the indicated concentrations of ORF2 (0.1, 0.3, and 1 μg). For all studies, luciferase assays were performed at 48 h after transfection using a dual-luciferase assay. A plasmid expressing Renilla luciferase under the control of a minimal herpesvirus TK promoter was used as an internal control for all samples. The results are the average of three independent experiments, and error bars denote standard errors. An asterisk indicates significant differences (P < 0.05) in promoter activity as determined by one-way analysis of variance and Fisher's least significant difference multiple-means comparison tests.

Article Snippet: Forty-eight hours after transfection cells were collected and processed for Western blot analysis using a Flag antibody (1:1,000 dilution; Sigma) to detect ORF2, a β-catenin antibody (1:5000 dilution), or an anti-V5 antibody (1:5,000 dilution; Rockland) to detect MAML1.

Techniques: Luciferase, Plasmid Preparation, Transfection, Expressing, Control, Activity Assay, Comparison

Journal: Journal of Virology

Article Title: β-Catenin, a Transcription Factor Activated by Canonical Wnt Signaling, Is Expressed in Sensory Neurons of Calves Latently Infected with Bovine Herpesvirus 1

doi: 10.1128/JVI.02971-15

Figure Lengend Snippet: Regulation of cell survival by ORF2, β-catenin, and MAML1 following growth factor withdrawal. (A) Neuro-2A cells were cotransfected with a plasmid expressing β-catenin alone, or with MAML1, a plasmid expressing ORF2, and a plasmid expressing the LacZ gene (transfection control). One microgram of each plasmid was used for transfection, and an empty plasmid was used to maintain equal concentrations of plasmid DNA for the respective transfections. Twenty-four hours after transfection, cells were seeded into new plates at a low density (4,000 cells/cm2) and then incubated with medium containing 0.5% serum for 3 days. Cells were fixed, and β-Gal+ cells were counted as described in Materials and Methods. (B) The number of β-Gal+ Neuro-2A cells transfected with the β-catenin expression plasmid (S33Y) was arbitrarily set at 1. The number of β-Gal+ Neuro-2A cells in the respective studies was normalized to the β-catenin level to calculate the fold of cell survival. The average of four independent experiments is shown. An asterisk denotes significant differences (P < 0.05) in β-Gal+ Neuro-2A cells containing β-catenin with MAML1 plus ORF2 relative to values with other plasmids or combinations, as determined by one-way analysis of variance and Fisher's least significant difference multiple-means comparison tests.

Article Snippet: Forty-eight hours after transfection cells were collected and processed for Western blot analysis using a Flag antibody (1:1,000 dilution; Sigma) to detect ORF2, a β-catenin antibody (1:5000 dilution), or an anti-V5 antibody (1:5,000 dilution; Rockland) to detect MAML1.

Techniques: Plasmid Preparation, Expressing, Transfection, Control, Incubation, Comparison

Journal: International Journal of Biological Sciences

Article Title: Disialyl GD2 ganglioside suppresses ICAM-1-mediated invasiveness in human breast cancer MDA-MB231 cells

doi: 10.7150/ijbs.16903

Figure Lengend Snippet: RT-PCR primer sequences

Article Snippet: Glyceraldehydes-3-phosphodehydrogenase (GAPDH) (MAB-8145) (Chemicon, USA), SARK/JNK(#9252), p-SARK/JNK(#9255) (Cell signaling, USA) and GD3 synthase (xw-8145) (ProSci incorporated, USA) were purchased.

Techniques:

Journal: International Journal of Biological Sciences

Article Title: Disialyl GD2 ganglioside suppresses ICAM-1-mediated invasiveness in human breast cancer MDA-MB231 cells

doi: 10.7150/ijbs.16903

Figure Lengend Snippet: Real-time PCR primer sequences

Article Snippet: Glyceraldehydes-3-phosphodehydrogenase (GAPDH) (MAB-8145) (Chemicon, USA), SARK/JNK(#9252), p-SARK/JNK(#9255) (Cell signaling, USA) and GD3 synthase (xw-8145) (ProSci incorporated, USA) were purchased.

Techniques: Real-time Polymerase Chain Reaction

Journal: International Journal of Biological Sciences

Article Title: Disialyl GD2 ganglioside suppresses ICAM-1-mediated invasiveness in human breast cancer MDA-MB231 cells

doi: 10.7150/ijbs.16903

Figure Lengend Snippet: A) GD3 synthase gene expression patterns in human breast cancer cells. Total mRNA was isolated from breast cancer and SK-Mel II melanoma cells. The bar graphs represent the relative band intensity obtained from RT-PCR results by densitometry. For the expression levels of GD3 synthase mRNA, RT-PCR (B) and real-time PCR (C) analyses were performed. The protein expression was analyzed by Western blot (D). The pc3-GD3s cells were cultured with 1 mg/ml G-418. All experiments have been performed at least three times and we examined mean differences between groups by using the error bar graph procedure.

Article Snippet: Glyceraldehydes-3-phosphodehydrogenase (GAPDH) (MAB-8145) (Chemicon, USA), SARK/JNK(#9252), p-SARK/JNK(#9255) (Cell signaling, USA) and GD3 synthase (xw-8145) (ProSci incorporated, USA) were purchased.

Techniques: Gene Expression, Isolation, Reverse Transcription Polymerase Chain Reaction, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Cell Culture

Journal: International Journal of Biological Sciences

Article Title: Disialyl GD2 ganglioside suppresses ICAM-1-mediated invasiveness in human breast cancer MDA-MB231 cells

doi: 10.7150/ijbs.16903

Figure Lengend Snippet: The pc3-GD3s cells show changes in morphology but not in cell growth Cell were cultured in DMEM supplemented with 10% or 5% FBS. The pc3 (◆ ) and pc3-GD3s cells (■) were plated in triplicates. Cell growth in different serum conditions was assessed by the XTT assay after 24 hr (A). The cell morphology was observed by phase contrast microscopy (B). Inhibition of invasiveness in GD3 synthase-transfected MDA-MB231 cells (C). Representative microphotographs of Hematoxylin and Eosin stained cells. Cells that invaded through the gelatin and were located on the underside of the filter were counted. All experiments performed at least three times and we examined mean differences between groups by using the error bar graph procedure.

Article Snippet: Glyceraldehydes-3-phosphodehydrogenase (GAPDH) (MAB-8145) (Chemicon, USA), SARK/JNK(#9252), p-SARK/JNK(#9255) (Cell signaling, USA) and GD3 synthase (xw-8145) (ProSci incorporated, USA) were purchased.

Techniques: Cell Culture, XTT Assay, Microscopy, Inhibition, Transfection, Staining

Journal: International Journal of Biological Sciences

Article Title: Disialyl GD2 ganglioside suppresses ICAM-1-mediated invasiveness in human breast cancer MDA-MB231 cells

doi: 10.7150/ijbs.16903

Figure Lengend Snippet: Differential regulation of ICAM-1 expression in pc3-GD3s-trasnfected SK-BR3 (ER-) and MCF-7 (ER+) cells. Expression levels of GM3 synthase, GD3 synthase and ICAM-1 mRNAs in the pc3-GD3s-transfected SK-BR3 (ER-) were checked by RT-PCR (A) and relative band intensities were compared with each β-actin control (B). β-Actin was included as an internal control. For MCF-7 (ER+) cells, ICAM-1 mRNA level in the pc3-GD3s-transfected MCF-7 cells was examined by Western blot analysis (C) and relative band intensity was also compared with GAPDH control (D). All experiments performed at least three times. We examined mean differences between groups by using the error bar graph procedure.

Article Snippet: Glyceraldehydes-3-phosphodehydrogenase (GAPDH) (MAB-8145) (Chemicon, USA), SARK/JNK(#9252), p-SARK/JNK(#9255) (Cell signaling, USA) and GD3 synthase (xw-8145) (ProSci incorporated, USA) were purchased.

Techniques: Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Control, Western Blot

Journal: International Journal of Molecular Medicine

Article Title: Long non-coding RNA FOXD2-AS1 regulates the tumorigenesis and progression of breast cancer via the S100 calcium binding protein A1/Hippo signaling pathway

doi: 10.3892/ijmm.2020.4699

Figure Lengend Snippet: Knockdown of FOXD2-AS1 suppresses cell proliferation, migration and invasion in breast cancer. (A and B) Following transfection with shRNA-FOXD2-AS, the mRNA level of FOXD2-AS1 was measured by RT-qPCR in MCF-7 and MDA-MB-468 cells. (C and D) CCK-8 assay was performed to determine cell proliferation following transfection. (E-G) Cell cycle distribution was determined and analyzed by FACS. (H and I) The protein expression of cyclin E1, CDK2 and p21 was determined by western blot analysis. Knockdown of FOXD2-AS1 suppresses cell proliferation, migration and invasion in breast cancer. (J) Wound healing assay was performed to detect the migration of both MCF-7 and MDA-MB-468 cells. (K and L) Relative migration rate of MCF-7 and MDA-MB-468 cells was quantified, respectively. (M) Transwell assay was performed to detect the invasion of both MCF-7 and MDA-MB-468 cells. (N and O) Relative cell invasive rate of MCF-7 and MDA-MB-468 cells was quantified, respectively. (P) MMP-2 and MMP-9 protein expression in MCF-7 transfected with shRNA-FOXD2-AS1 was detected and quantified. (Q) MMP-2 and MMP-9 protein expression in MDA-MB-468 transfected with shRNA-FOXD2-AS1 was detected and quantified. * P<0.05, **P<0.01 and *** P<0.001, vs. shRNA-NC. CDK2, cyclin-dependent kinase-2; MMP, matrix metalloproteinase.

Article Snippet: To overexpress FOXD2-AS1, an overexpression vector (pcDNA FOXD2-AS1) and its corresponding negative control vector (pcDNA-NC) were synthesized by Shanghai GenePharma Co., Ltd. Short hairpin (sh)RNAs targeting FOXD2-AS1 (100 nM; shRNA-FOXD2-AS1-1 and shRNA-FOXD2-AS1-2) and a negative scramble control shRNA (shRNA) (also purchased from Shanghai GenePharma Co., Ltd.) were used to knock down FOXD2-AS1 expression.

Techniques: Knockdown, Migration, Transfection, shRNA, Quantitative RT-PCR, CCK-8 Assay, Expressing, Western Blot, Wound Healing Assay, Transwell Assay

Journal: International Journal of Molecular Medicine

Article Title: Long non-coding RNA FOXD2-AS1 regulates the tumorigenesis and progression of breast cancer via the S100 calcium binding protein A1/Hippo signaling pathway

doi: 10.3892/ijmm.2020.4699

Figure Lengend Snippet: Knockdown of FOXD2-AS1 regulates the Hippo/YAP signaling pathway. (A) The protein expression of Hippo/YAP signaling pathway-related genes (p-YAP, YAP, p-LATS1, LATS1, MST1 and MST2) was determined by western blot analysis in MCF-7 cells. (B) The protein expression of Hippo/YAP signaling pathway-related genes (p-YAP, YAP, p-LATS1, LATS1, MST1 and MST2) was determined by western blot analysis in MDA-MB-468 cells. * P<0.05, ** P<0.01 and *** P<0.001, vs. shRNA-NC. LATS1, large tumor suppressor homolog 1; MST, mammalian STE20-like protein kinase; YAP, yes-associated protein.

Article Snippet: To overexpress FOXD2-AS1, an overexpression vector (pcDNA FOXD2-AS1) and its corresponding negative control vector (pcDNA-NC) were synthesized by Shanghai GenePharma Co., Ltd. Short hairpin (sh)RNAs targeting FOXD2-AS1 (100 nM; shRNA-FOXD2-AS1-1 and shRNA-FOXD2-AS1-2) and a negative scramble control shRNA (shRNA) (also purchased from Shanghai GenePharma Co., Ltd.) were used to knock down FOXD2-AS1 expression.

Techniques: Knockdown, Expressing, Western Blot, shRNA

Journal: International Journal of Molecular Medicine

Article Title: Long non-coding RNA FOXD2-AS1 regulates the tumorigenesis and progression of breast cancer via the S100 calcium binding protein A1/Hippo signaling pathway

doi: 10.3892/ijmm.2020.4699

Figure Lengend Snippet: S100A1 is upregulated in breast cancer cells. (A) The mRNA level of S100A1 in MCF-10A and human breast cancer cell lines (MCF-7, MDA-MB-468, MDA-MB-453 and BT-549) was determined by RT-qPCR. * P<0.05, ** P<0.01 and *** P<0.001, vs. MCF-10A cells. (B and C) In shRNA-FOXD2-AS1-trans-fected MCF-7 and MDA-MB-468 cells, the protein expression of Sl00A1 was detected by western blot analysis. * P<0.05 and *** P<0.001, vs. shRNA-NC. (D and E) Following transfection with pcDNA-FOXD2-AS1, the mRNA level of FOXD2-AS1 in MCF-7 and MDA-MB-468 cells was detected by RT-qPCR. ** P<0.01 and *** P<0.001, vs. pcDNA-NC. MCF-7 and MDA-MB-468 cells were transfected with shRNA-S100A1, and the protein expression and mRNA level of S100A1 in (F and G) MCF-7 and (H and I) MDA-MB-468 cells were determined by western blot analysis and RT-qPCR, respectively. ** P<0.01 and *** P<0.001, vs. shRNA-NC. S100A1, S100 calcium binding protein A1.

Article Snippet: To overexpress FOXD2-AS1, an overexpression vector (pcDNA FOXD2-AS1) and its corresponding negative control vector (pcDNA-NC) were synthesized by Shanghai GenePharma Co., Ltd. Short hairpin (sh)RNAs targeting FOXD2-AS1 (100 nM; shRNA-FOXD2-AS1-1 and shRNA-FOXD2-AS1-2) and a negative scramble control shRNA (shRNA) (also purchased from Shanghai GenePharma Co., Ltd.) were used to knock down FOXD2-AS1 expression.

Techniques: Quantitative RT-PCR, shRNA, Expressing, Western Blot, Transfection, Binding Assay

Journal: International Journal of Molecular Medicine

Article Title: Long non-coding RNA FOXD2-AS1 regulates the tumorigenesis and progression of breast cancer via the S100 calcium binding protein A1/Hippo signaling pathway

doi: 10.3892/ijmm.2020.4699

Figure Lengend Snippet: S100A1 mediates FOXD2-AS1-induced cell proliferation, migration and invasion in breast cancer. Following transfection with pcDNA-FOXD2-AS1, cells were transfected with shRNA-S100A1. (A and B) CCK-8 assay was performed to determine the proliferation of the differently treated cells. (C) Migratory ability of MCF-7 cells in the different treatment groups was determined by wound healing assay. (D) Invasive ability of MCF-7 cells in the different treatment groups was determined by Transwell assay. (E) Migratory ability of MDA-MB-468 cells in the different treatment groups was determined by wound healing assay. (F) Invasive ability of MDA-MB-468 cells in the different treatment groups was determined by Transwell assay. *** P<0.001, vs. pcDNA-NC; ## P<0.01 and ### P<0.001, vs. FOXD2-AS1 + shRNA-NC. S100A1, S100 calcium binding protein A1.

Article Snippet: To overexpress FOXD2-AS1, an overexpression vector (pcDNA FOXD2-AS1) and its corresponding negative control vector (pcDNA-NC) were synthesized by Shanghai GenePharma Co., Ltd. Short hairpin (sh)RNAs targeting FOXD2-AS1 (100 nM; shRNA-FOXD2-AS1-1 and shRNA-FOXD2-AS1-2) and a negative scramble control shRNA (shRNA) (also purchased from Shanghai GenePharma Co., Ltd.) were used to knock down FOXD2-AS1 expression.

Techniques: Migration, Transfection, shRNA, CCK-8 Assay, Wound Healing Assay, Transwell Assay, Binding Assay

Journal: International Journal of Molecular Medicine

Article Title: Long non-coding RNA FOXD2-AS1 regulates the tumorigenesis and progression of breast cancer via the S100 calcium binding protein A1/Hippo signaling pathway

doi: 10.3892/ijmm.2020.4699

Figure Lengend Snippet: Overexpression of LATS1 inhibits FOXD2-AS-induced cell proliferation, migration and invasion. (A and B) mRNA level of LATS1 in MCF-7 and MDA-MB-468 cells was detected by RT-qPCR in LATS1-overexpressing cells. *** P<0.001, vs. overexpression-NC (oe-NC). Following transfection with pcDNA-FOXD2-AS1, cells were transfected with pcDNA-LATS1 to overexpress LATS1. (C and D) CCK-8 assay was performed to determine the proliferation of the differently treated cells. (E) Migratory ability of MCF-7 cells in the different treatment groups was determined by wound healing assay. (F) Invasive ability of MCF-7 cells in the different treatment groups was determined by Transwell assay. (G) Migratory ability of MDA-MB-468 cells in the different treatment groups was determined by wound healing assay. (H) Invasive ability of MDA-MB-468 cells in the different treatment groups was determined by Transwell assay. *** P<0.001, vs. pcDNA-NC; ### P<0.001, vs. FOXD2-AS1 + shRNA-NC. LATS1, large tumor suppressor homolog 1.

Article Snippet: To overexpress FOXD2-AS1, an overexpression vector (pcDNA FOXD2-AS1) and its corresponding negative control vector (pcDNA-NC) were synthesized by Shanghai GenePharma Co., Ltd. Short hairpin (sh)RNAs targeting FOXD2-AS1 (100 nM; shRNA-FOXD2-AS1-1 and shRNA-FOXD2-AS1-2) and a negative scramble control shRNA (shRNA) (also purchased from Shanghai GenePharma Co., Ltd.) were used to knock down FOXD2-AS1 expression.

Techniques: Over Expression, Migration, Quantitative RT-PCR, Transfection, CCK-8 Assay, Wound Healing Assay, Transwell Assay, shRNA

Journal: International Journal of Molecular Medicine

Article Title: Long non-coding RNA FOXD2-AS1 regulates the tumorigenesis and progression of breast cancer via the S100 calcium binding protein A1/Hippo signaling pathway

doi: 10.3892/ijmm.2020.4699

Figure Lengend Snippet: Knockdown of FOXD2-AS1 suppresses tumor progression of breast cancer in vivo . (A) Each mouse was injected with 1.0×10 7 MDA-MB-468 cells, which were stably transfected with either shRNA negative control or shRNA-FOXD2-AS1, subcutaneously at the right axillary lymph node. Following sacrifice, the tumors were excised and photographed. (B and C) During the experiment, the mouse weight and tumor volume (tumor volume =1/2 × length × width 2 ) was monitored every 5 days until the mice were sacrificed. (D) Protein expression levels of p-YAP, YAP, p-LATS1 and LATS1 in the extracted tumors were determined by western blot analysis and were then quantified. *** P<0.001, vs. shRNA-NC. LATS1, large tumor suppressor homolog 1; YAP, yes-associated protein.

Article Snippet: To overexpress FOXD2-AS1, an overexpression vector (pcDNA FOXD2-AS1) and its corresponding negative control vector (pcDNA-NC) were synthesized by Shanghai GenePharma Co., Ltd. Short hairpin (sh)RNAs targeting FOXD2-AS1 (100 nM; shRNA-FOXD2-AS1-1 and shRNA-FOXD2-AS1-2) and a negative scramble control shRNA (shRNA) (also purchased from Shanghai GenePharma Co., Ltd.) were used to knock down FOXD2-AS1 expression.

Techniques: Knockdown, In Vivo, Injection, Stable Transfection, Transfection, shRNA, Negative Control, Expressing, Western Blot