Journal: Cancers

Article Title: Galactosylceramide Upregulates the Expression of the BCL2 Gene and Downregulates the Expression of TNFRSF1B and TNFRSF9 Genes, Acting as an Anti-Apoptotic Molecule in Breast Cancer Cells

doi: 10.3390/cancers16020389

Figure Lengend Snippet: Characteristics of murine mammary carcinoma 4T1 cells overexpressing murine UGT8a and GalCer. ( A ) Expression of UGT8a protein in parental 4T1 cells, control 4T1 cells transduced with vector alone (4T1.C), and 4T1 cells overexpressing UGT8a (4T1.UGT8a); ( B ) immunostaining of neutral GSLs from 4T1, 4T1.C, and 4T1.UGTa cells. Western blotting with anti-human UGT8 antibodies was used to analyze the expression of UGT8a. Cellular proteins (40 µg) separated by SDS-PAGE on 12% gel were transferred onto a nitrocellulose membrane. GAPDH was used as the internal control For GalCer detection, samples of neutral GSLs purified from 1 × 10 7 cells were applied to an HP-TLC plate. ( C ) Viability of 4T1.C and 4T1.UGT8a cells overexpressing UGT8a and GalCer incubated with increased concentrations of DOX for 48 h. The percentage of viable cells was determined using an MTT assay. Data represent the mean ± SD of six replicates from two independent measurements. Statistically significant differences (* p < 0.1). ( D ) Impact of intravenous administration of DOX on the growth of 4T1.C and 4T1.UGT8a tumors in BALB/c mice. 4T1.UGT8a—mice with 4T1.UGT8a tumors overexpressing UGT8a and GalCer treated with placebo (PBS); 4T1.UGT8 + DOX—mice with 4T1.UGT8a tumors treated with DOX; 4T1.C—mice with control 4T1.C tumors treated with placebo (PBS); 4T1.C + DOX—mice with 4T1.C tumours treated with DOX. Data was presented as mean tumor volume (±SD) for the group of mice ( n = 5) at the specified time intervals. Data was analyzed using the Prism 5.0 two-way ANOVA Dunnett’s post-test (* p < 0.05). The original western blot of A,B is in .

Article Snippet: Using PCR, human UGT8 cDNA was generated from the MDA-MB-231 cDNA library as a template for the primers forXhoI-UGT8 and revBamHI-UGT8 , and mouse galactosylceramide synthase (UGT8a) cDNA was amplified from the pCMV6-Entry vector containing UGT8a cDNA (MR208632, 1626 bp; OriGene, Rockville, MD, USA) with forEcoRI-UGT8a and revMluI-UGT8a primers ( ).

Techniques: Expressing, Control, Transduction, Plasmid Preparation, Immunostaining, Western Blot, SDS Page, Membrane, Purification, Incubation, MTT Assay

Journal: British Journal of Cancer

Article Title: Ceramide galactosyltransferase (UGT8) is a molecular marker of breast cancer malignancy and lung metastases

doi: 10.1038/sj.bjc.6605750

Figure Lengend Snippet: Immunohistochemical staining of primary breast carcinoma ( A ) and paired lung metastasis ( B ) with anti-UGT8 rabbit polyclonal antibodies.

Article Snippet: UGT8 was detected with rabbit polyclonal antibodies (Atlas Antibodies, Stockholm, Sweden) and horseradish peroxidase-conjugated donkey anti-rabbit immunoglobulins (Jackson ImmunoResearch, West Grove, PA, USA).

Techniques: Immunohistochemical staining, Staining

Journal: British Journal of Cancer

Article Title: Ceramide galactosyltransferase (UGT8) is a molecular marker of breast cancer malignancy and lung metastases

doi: 10.1038/sj.bjc.6605750

Figure Lengend Snippet: Immunohistochemical staining of primary breast carcinomas representing different malignancy grades (G1–G3). UGT8 expression in primary breast carcinomas of different malignancy grades: ( A ) G1 ( n =9), ( B ) G2 ( n =10), and ( C ) G3 ( n =21). Reaction intensities with rabbit polyclonal antibodies for UGT8 ( D ) were calculated on the basis of the semi-quantitative IRS scale of and are represented as means; * P <0.01 for primary breast tumours of grade G3 as compared with primary breast tumour of grade G2, and # P <0.001 for primary breast tumours of grade G3 as compared with primary breast tumours of grade G1 (Mann–Whitney U -test).

Article Snippet: UGT8 was detected with rabbit polyclonal antibodies (Atlas Antibodies, Stockholm, Sweden) and horseradish peroxidase-conjugated donkey anti-rabbit immunoglobulins (Jackson ImmunoResearch, West Grove, PA, USA).

Techniques: Immunohistochemical staining, Staining, Expressing, MANN-WHITNEY

Journal: British Journal of Cancer

Article Title: Ceramide galactosyltransferase (UGT8) is a molecular marker of breast cancer malignancy and lung metastases

doi: 10.1038/sj.bjc.6605750

Figure Lengend Snippet: UGT8 expression (A) in node-negative invasive ductal carcinomas ( n =22) and (B) node-positive invasive ductal carcinomas ( n =18). P <0.05 for UGT8-expressing, node-negative primary breast tumours as compared with node-positive primary tumours (Mann–Whitney U -test). Reaction intensities with rabbit polyclonal antibodies for UGT8 were calculated on the basis of the semi-quantitative IRS scale of and are represented as means.

Article Snippet: UGT8 was detected with rabbit polyclonal antibodies (Atlas Antibodies, Stockholm, Sweden) and horseradish peroxidase-conjugated donkey anti-rabbit immunoglobulins (Jackson ImmunoResearch, West Grove, PA, USA).

Techniques: Expressing, MANN-WHITNEY

Journal: British Journal of Cancer

Article Title: Ceramide galactosyltransferase (UGT8) is a molecular marker of breast cancer malignancy and lung metastases

doi: 10.1038/sj.bjc.6605750

Figure Lengend Snippet: ( A ) Western blot analysis of anti-UGT8 rabbit polyclonal antibodies binding to cellular proteins of breast cancer cell lines. Cell lysates, equivalent to 15 μ g of protein, were separated by SDS-PAGE under reducing conditions on an 8% gel and electrophoretically transferred onto a nitrocellulose membrane. β -Actin served as an internal control. ( B ) Expression of UGT8 mRNA in breast cancer cell lines. Real-time RT-PCR was used to analyse UGT8 mRNA. UGT8 levels were normalised against GAPDH and cell line MCF-7 was assigned as a calibrator sample. Results are expressed as means.

Article Snippet: UGT8 was detected with rabbit polyclonal antibodies (Atlas Antibodies, Stockholm, Sweden) and horseradish peroxidase-conjugated donkey anti-rabbit immunoglobulins (Jackson ImmunoResearch, West Grove, PA, USA).

Techniques: Western Blot, Binding Assay, SDS Page, Membrane, Control, Expressing, Quantitative RT-PCR

Journal: British Journal of Cancer

Article Title: Ceramide galactosyltransferase (UGT8) is a molecular marker of breast cancer malignancy and lung metastases

doi: 10.1038/sj.bjc.6605750

Figure Lengend Snippet: Immunostaining of neutral glycolipids from human breast cancer cell lines, separated by HP-TLC, with anti-GalCer rabbit polyclonal antibodies. For the analysed cell lines, an aliquot of total neutral glycolipids corresponding to 1 × 10 7 cells was applied to the HP-TLC plate.

Article Snippet: UGT8 was detected with rabbit polyclonal antibodies (Atlas Antibodies, Stockholm, Sweden) and horseradish peroxidase-conjugated donkey anti-rabbit immunoglobulins (Jackson ImmunoResearch, West Grove, PA, USA).

Techniques: Immunostaining

Journal: PLoS ONE

Article Title: Human iPSC-derived astrocytes generated from donors with globoid cell leukodystrophy display phenotypes associated with disease

doi: 10.1371/journal.pone.0271360

Figure Lengend Snippet: (A) A simplified schematic of the glycosphingolipid pathway examined in the current study and the genes encoding the enzymes responsible for their biosynthesis. The gene encoding galactosylceramidase ( GALC ), which is defective in Krabbe disease, is indicated in red. (B) Astrocytes generated from the Krabbe donors had significantly elevated levels of psychosine. (C) No significant difference in total non-hydroxylated galactosylceramide was observed between control and Krabbe astrocytes. (D) Increased total non-hydroxylated glucosylceramide was observed in Krabbe astrocytes. (E-H) Compensatory changes in the expression of genes involved in the local glycosphingolipid biosynthesis pathway. For the galactosylceramide arm, a significant downregulation of genes encoding ceramide galactosyltransferase ( UGT8 ) and acid ceramidase ( ASAH1 ), which are responsible for the biosynthesis of galactosylceramide and psychosine, respectively, was observed in Krabbe astrocytes compared to controls. For the glucosylceramide arm, an upregulation of ceramide glucosyltransferase ( UGCG ) and downregulation of glucocerebrosidase 1 ( GBA1 ) was observed in Krabbe astrocytes compared to controls. ** p < 0.01, *** p < 0.001, **** p < 0.0001. Data for individual donor subjects is shown as a box and whisker plot with median represented and errors bars indicating min/max values. Statistical analysis was conducted using t-tests on pooled data comparing control vs. Krabbe cells. N.D. = not detected.

Article Snippet: The following TaqMan probes were used in our study (all from Thermo-Fisher Scientific): FAM-labeled IL6 (Hs00174131_m1), IL8 ( CXCL8 , Hs00174103_m1), IL1B (Hs01555410_m1), CCL2 (MCP-1, Hs00234140_m1), ASAH1 (Hs00602774_m1), UGT8 (Hs00409958_m1), UGCG (Hs00916612_m1), GBA (GBA1, Hs00986836_g1), RIPK3 (Hs00179132_m1), and MT1E (Hs01652848_g1).

Techniques: Generated, Control, Expressing, Whisker Assay

Journal: Cell & Bioscience

Article Title: Activation of goblet cell Piezo1 alleviates mucus barrier damage in mice exposed to WAS by inhibiting H3K9me3 modification

doi: 10.1186/s13578-023-00952-5

Figure Lengend Snippet: Impaired mucus barrier function in the WAS model mice. A , B Pain threshold and AWR score in the control and WAS mice (n ≥ 6). C , D Colon mucus thickness and increased mucus thickness in the control and WAS mice measured by horizontal ussing chamber (n ≥ 6). E RT-PCR of mucin2 mRNA in the mucosa of control and WAS mice (n ≥ 6). F The ratio of goblet cells to epithelial cells in the control and WAS mice (n ≥ 6). G Immunofluorescence of mucin2 in the control and WAS mice; bar length: 50 μm. H Alcian staining of control and WAS mice colon; bar length: 50 μm. I FISH of control and WAS mice colon, mucus was stained with UEA1 (green), bacteria were detected by fluorescence in situ hybridization with general bacterial 16S probes EUB (red) and DNA stained using DAPI (blue); bar length: 50 μm and 20 μm

Article Snippet: As in previous research [ ], paraffin-embedded Carnoy’s-fixed sections were hybridized with 10 ng/ml of a general bacterial 16S rRNA probe (EUB338, red, 5′-GCT GCC TCC CGT AGG AGT-3′, Boster) and immunostained for Mucin2 using the UEA-1 (Bauhinia bean lectin, FL-1061-2, Vector labs).

Techniques: Control, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Staining, Bacteria, Fluorescence, In Situ Hybridization

Journal: Cell & Bioscience

Article Title: Activation of goblet cell Piezo1 alleviates mucus barrier damage in mice exposed to WAS by inhibiting H3K9me3 modification

doi: 10.1186/s13578-023-00952-5

Figure Lengend Snippet: Piezo1 agonist improved mucus barrier function and ameliorated intestinal motility disorder in WAS mice. A , B Pain threshold and AWR score in the control, WAS and WAS + Yoda1 mice (n ≥ 5). C , D Colon mucus thickness and increased mucus thickness in the control, WAS and WAS + Yoda1 mice measured by horizontal Ussing chamber (n ≥ 5). E Alcian staining of control, WAS and WAS + Yoda1 mice colon; bar length: 50 μm. F The ratio of goblet cells to epithelial cells in the control, WAS and WAS + Yoda1 mice (n ≥ 6). G RT-PCR of mucin2 mRNA in the mucosa of control, WAS and WAS + Yoda1 mice (n ≥ 5). H FISH of control, WAS and WAS + Yoda1 mice colon; mucus was stained with UEA1 (green), bacteria were detected by fluorescence in situ hybridization with general bacterial 16S probes EUB (red) and DNA stained using DAPI (blue); bar length: 50 μm and 20 μm. I Results of Ink propulsion test in the control, WAS and WAS + Yoda1 mice. J – L Spontaneous activities and Acetylcholine (Ach) induced responses of colonic longitudinal muscles, including MI and MI compared to baseline, in the control, WAS and WAS + Yoda1 mice (n ≥ 5)

Article Snippet: As in previous research [ ], paraffin-embedded Carnoy’s-fixed sections were hybridized with 10 ng/ml of a general bacterial 16S rRNA probe (EUB338, red, 5′-GCT GCC TCC CGT AGG AGT-3′, Boster) and immunostained for Mucin2 using the UEA-1 (Bauhinia bean lectin, FL-1061-2, Vector labs).

Techniques: Control, Staining, Reverse Transcription Polymerase Chain Reaction, Bacteria, Fluorescence, In Situ Hybridization, Muscles

Journal: Cell & Bioscience

Article Title: Activation of goblet cell Piezo1 alleviates mucus barrier damage in mice exposed to WAS by inhibiting H3K9me3 modification

doi: 10.1186/s13578-023-00952-5

Figure Lengend Snippet: Decreased mucus expression and increased methylation in the Piezo1 flox-mucin2 Cre mice. A Immunohistochemistry of Piezo1 in WT and Piezo1 flox-mucin2 Cre mice (KO mice); bar length: 20 μm. B Western blot of Piezo1 in WT and KO mice. C , D Alcian staining and statistical analysis of mucus thickness in WT and KO mice (n ≥ 6); bar length: 50 μm. E RT-PCR of mucin2 in WT and KO mice (n ≥ 6). F , G Immunofluorescence and statistical analysis of goblet cells/ epithelial cells in WT and KO mice (n ≥ 6); bar length: 20 μm. H FISH of WT and KO mice, mucus was stained with UEA1 (green), bacteria were detected by fluorescence in situ hybridization with general bacterial 16S probes EUB (red) and DNA stained using DAPI (blue); bar length: 20 μm. I – J Western blot and statistical analysis of SUV39h1, Hdac3 and H3K9me3 in the colon mucosa of WT and KO mice. K Immunofluorescence for co-localization of AGR2 (label of goblet cells) and Piezo1 in intestinal mucosa of WT and KO mice; bar length: 20 μm

Article Snippet: As in previous research [ ], paraffin-embedded Carnoy’s-fixed sections were hybridized with 10 ng/ml of a general bacterial 16S rRNA probe (EUB338, red, 5′-GCT GCC TCC CGT AGG AGT-3′, Boster) and immunostained for Mucin2 using the UEA-1 (Bauhinia bean lectin, FL-1061-2, Vector labs).

Techniques: Expressing, Methylation, Immunohistochemistry, Western Blot, Staining, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Bacteria, Fluorescence, In Situ Hybridization

Journal: PLoS ONE

Article Title: Microbubble-based enhancement of radiation effect: Role of cell membrane ceramide metabolism

doi: 10.1371/journal.pone.0181951

Figure Lengend Snippet: Transfected cells were identified by the fluorescence of GFP. A) Transient transfection, showing fewer transfected cells than in the stably transfected cells. The levels of the UGT8 gene and the UGT8 protein were evaluated after the stable transfection using RT-PCR, and ELISA, which verified the change in the levels of regulation. B) Ceramide trafficking in live images of cultured PC3 cells using BODIPY TR-labeled C 5 ceramide (red fluorescence), nuclear position is indicated by the nucleic acid stain Hoechst 33258 (blue fluorescence) with green fluorescence indicating transfection of constructs. After 3 h, control and down regulated cells demonstrtaed a higher level of ceramide uptake (red) that masks the green fluorescence compared to the cells over expressing UGT8. After 24 h, control and down regulated cells retained the exogenous ceramide (red) contrary to the cells over expressing UGT8 . Scale bars represent 50 μm.

Article Snippet: Supernatants were then assayed with UGT8 detection carried out using an ELISA kit (Cat# MBS053427 from MyBiosource, San Diego, CA), following the manufacturer's instructions.

Techniques: Transfection, Fluorescence, Stable Transfection, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Cell Culture, Labeling, Staining, Construct, Expressing

Journal: PLoS ONE

Article Title: Microbubble-based enhancement of radiation effect: Role of cell membrane ceramide metabolism

doi: 10.1371/journal.pone.0181951

Figure Lengend Snippet: A) Control cells demonstrating increased levels of ceramide stained under various treatment conditions (brown, arrow). More condensed brown labelling was observed in down regulated cells under various treatments (arrow). In contrast, cells with up regulated UGT8, that were also subjected to similar conditions, demonstrate less ceramide labeling (arrows). B) UGT8 labelling was most intense in the up-regulated cells compared to the control or to the down-regulated cells (arrows). Scale bar represents 25 μm. C) Clonogenic assays indicate that control and up-regulated cells were less susceptible to treatment when compared to cells stably transfected with UGT8 inhibitory shRNA.

Article Snippet: Supernatants were then assayed with UGT8 detection carried out using an ELISA kit (Cat# MBS053427 from MyBiosource, San Diego, CA), following the manufacturer's instructions.

Techniques: Staining, Labeling, Stable Transfection, Transfection, shRNA

Journal: PLoS ONE

Article Title: Microbubble-based enhancement of radiation effect: Role of cell membrane ceramide metabolism

doi: 10.1371/journal.pone.0181951

Figure Lengend Snippet: The upper two panels show low magnification whole tumour sections and the lower two panels illustrate high magnification images. Controls, not transfected, or transfected with scrambled shRNA (sham) demonstrated apoptotic cell death more with combined treatments and less with single treatments. However, tumours induced by cells stably transfected with anti- UGT8 sh-RNA, show different types of cell death and fibrosis. Cell death was less obvious in xenograft tumours initiated from cells with up-regulated UGT8 . Scale bar for low magnification images is 1mm and for high magnification represents 50 μm.

Article Snippet: Supernatants were then assayed with UGT8 detection carried out using an ELISA kit (Cat# MBS053427 from MyBiosource, San Diego, CA), following the manufacturer's instructions.

Techniques: Transfection, shRNA, Stable Transfection

Journal: PLoS ONE

Article Title: Microbubble-based enhancement of radiation effect: Role of cell membrane ceramide metabolism

doi: 10.1371/journal.pone.0181951

Figure Lengend Snippet: Control and sham tumours illustrate an increase in levels of apoptotic cell death which was significant in the combined treatments. A further increase in cell death is observed in tumours induced by cells stably transfected with anti- UGT8 sh-RNA. In contrast, less cell death is observed with up-regulated UGT8 . Scale bar represents 50 μm.

Article Snippet: Supernatants were then assayed with UGT8 detection carried out using an ELISA kit (Cat# MBS053427 from MyBiosource, San Diego, CA), following the manufacturer's instructions.

Techniques: Stable Transfection, Transfection

Journal: PLoS ONE

Article Title: Microbubble-based enhancement of radiation effect: Role of cell membrane ceramide metabolism

doi: 10.1371/journal.pone.0181951

Figure Lengend Snippet: Control and sham tumours illustrate an increase in levels of ceramide which is significant in the combined treatments (dark red). An increase in ceramide is observed in tumours induced by cells stably transfected with anti- UGT8 sh-RNA. Less ceramide is observed with up-regulated UGT8 . Magnification bar represents 20 μm.

Article Snippet: Supernatants were then assayed with UGT8 detection carried out using an ELISA kit (Cat# MBS053427 from MyBiosource, San Diego, CA), following the manufacturer's instructions.

Techniques: Stable Transfection, Transfection

Journal: PLoS ONE

Article Title: Microbubble-based enhancement of radiation effect: Role of cell membrane ceramide metabolism

doi: 10.1371/journal.pone.0181951

Figure Lengend Snippet: When UGT8 levels are reduced, ceramide, either produced at the cellular membrane or in the endoplasmic reticulum, accumulates or initiates an apoptotic signal. However, when UGT8 levels are elevated, it adds a sugar moiety to ceramide in the Golgi apparatus, converting it to galactosylceramide, and this leads to the reduction of ceramide levels and possibly its apoptotic signalling, thus making cells more resistant to therapy.

Article Snippet: Supernatants were then assayed with UGT8 detection carried out using an ELISA kit (Cat# MBS053427 from MyBiosource, San Diego, CA), following the manufacturer's instructions.

Techniques: Produced