facs sc 5279 tra 1 60 r d systems mouse monoclonal  (R&D Systems)

Journal: EMBO Reports

Article Title: Terminal α1,2-fucosylation of glycosphingolipids by FUT1 is a key regulator in early cell-fate decisions

doi: 10.1038/s44319-024-00243-1

Figure Lengend Snippet: ( A ) Up at left: Quantification of pluripotent gene expression as measured by qPCR. Up at right: Quantification of the percentage of hESCs and hESCs-derived DE on day 8 showing the expression of OCT3/4 in histograms, bar chart, and MFI relative to hESCs. Bottom: Quantification of the percentage of hESCs and hESCs-derived DE on day 8 showing the expression of NANOG in histograms, bar chart, and MFI relative to hESCs. n = 4 technical replicates. ( B ) Up and bottom at left: Quantification of DE-specific markers, FOXA2 and SOX17 as measured by qPCR after hESC differentiation into DE using protocol 1, and FOXA2, SOX17, and HHEX as measured by qPCR after hESC differentiation into DE using protocol 2 ( n = 3 technical replicates). Bottom at right: Quantification of the percentage of hESCs and hESCs-derived DE on day 8 showing the expression of α-FP in histograms and bar chart and MFI relative to hESCs. ( C ) Up at left: Schematic illustration of hESC differentiation into NPCs via bFGF and RA signaling and BMP inhibition over 6 d. Up at right: Representative immunofluorescent cultures illustrate day 6 NPCs expressing OTX2 (green) and PAX6 (red). Nuclei are stained with DAPI (blue). Scale bars represent 100 μm. Bottom: qRT-PCR and flow cytometry analyses of OCT3/4, PAX6, SOX1. The expression of FUT1 and α-fucose in iPSCs during pluripotency and differentiation is identical to that of hESCs ( D ) Quantification of the percent of positive cells expressing both the FT1 and OCT3/4 proteins and MFI in hESCs, a pool of hiPSCs and a pool of human fibroblasts. Pooled sample, n = 3 cell lines. n = 3 technical replicates. ( E ) Quantification of the percent of positive cells expressing FT1 protein and α-fucose residues and MFI in hESCs, a pool of hiPSCs, and a pool of human fibroblasts. Pooled sample, n = 3 cell lines. n = 3 technical replicates. Data information: In qPCR ( A , B ), data are presented as means ± SD. Two-tailed Student’s t-tests * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. In FACS ( A – E ), data are presented as means ± SD. Ordinary One-way ANOVA ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: NKX2.5 , Santa Cruz Biotechnology (mouse, monoclonal) (FACS) , , sc-376565.

Techniques: Gene Expression, Derivative Assay, Expressing, Inhibition, Staining, Quantitative RT-PCR, Flow Cytometry, Two Tailed Test

Journal: EMBO Reports

Article Title: Terminal α1,2-fucosylation of glycosphingolipids by FUT1 is a key regulator in early cell-fate decisions

doi: 10.1038/s44319-024-00243-1

Figure Lengend Snippet: ( A ) Top at left: Quantification of the percent of positive cells expressing α-fucose residues during pluripotency in three lines of hESCs: WA09, WIBR1, and WIBR2, as measured in FACS. Top at center: Quantification of the percent of positive cells expressing FT1 protein and MFI of WIBR1 and WIBR2 hESCs 30 h after FUT1 mRNA silencing (siFUT1). Negative control is WIBR1 and WIBR2 hESCs transfected with non-targeting siRNA (siNT). Top at right: Quantification of the gene expression of FUT1 in WT, siNT, and siFUT1, WA09 hESCs 30 h after FUT1 mRNA silencing, as measured by qPCR, and representative histograms showing relative counts of WT, siNT, and siFUT1, WA09 hESCs expressing α-fucose residues 54, 78, and 102 h after FUT1 mRNA silencing. Bottom: MFI of WT, siNT, and siFUT1, WA09 hESCs expressing α-fucose residues 54, 78, and 102 h after FUT1 mRNA silencing. ( B ) Schematic of Globo-H and SSEA-5 structures encompassing the α1,2-fucose end residue with corresponding GTs. ( C ) Top: Representative histograms showing relative counts of WT, siNT, and siFUT1, WA09 hESCs expressing SSEA-5 and MFI 54, 78, and 102 h after FUT1 mRNA silencing. Bottom: Representative histograms showing relative counts of WT, siNT, and siFUT1, WA09 hESCs expressing Globo-H and MFI 54, 78, and 102 h after FUT1 mRNA silencing. ( D ) Quantification of the gene expression of FUT2 in WT, siNT, and siFUT1, WA09 hESCs 30, 54, and 78 h after FUT1 mRNA silencing, as measured by qPCR. ( E ) Representative histograms showing relative counts of WT, siNT, and siFUT1, WA09 hESCs expressing HOPX and MFI 54, 78, and 102 h after silencing. n = 4 technical replicates. Data information: In ( A , C , E ), data are presented as means ± SD. Ordinary One-way ANOVA * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 or non-significant (NS). In ( D ), data are presented as means ± SEM. Two-tailed Student’s t-tests * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: NKX2.5 , Santa Cruz Biotechnology (mouse, monoclonal) (FACS) , , sc-376565.

Techniques: Expressing, Negative Control, Transfection, Gene Expression, Residue, Two Tailed Test

Journal: EMBO Reports

Article Title: Terminal α1,2-fucosylation of glycosphingolipids by FUT1 is a key regulator in early cell-fate decisions

doi: 10.1038/s44319-024-00243-1

Figure Lengend Snippet: ( A ) Left: Representative histograms showing counts and MFI of FUT1 positive hESCs (FUT1 + ECs) expressing Globo-H compared to WT hESCs. Center: Representative images showing hESC nuclei (blue) and Globo-H expression (green). Right: Representative histograms showing counts and MFI of FUT1 + ECs expressing SSEA-5 compared to hESCs. n = 3 biological replicates. ( B ) Left: Pluripotent NANOG, SOX2, and OCT3/4 mRNA expression levels in WT hESCs, control hESCs (control) and FUT1 + ECs, three passages after mock and FUT1 transfection, respectively. n = 3 clones for each clone n = 2 technical replicates. Right top: Representative bright-field and endogenous fluorescence protein (green) images of control colony morphology after one passage in culture, on day 3. Right bottom: Representative bright-field and endogenous fluorescence protein (green) images of FUT1 + EC colony morphology after one passage in culture, on day 3. n = 2 technical replicates. ( C ) mRNA expression levels of pluripotent OCT3/4 and mesoderm-specific markers, MESP1 and BRY, in WT hESCs, control, and FUT1 + ECs, on day 0 and after differentiation into LM for 1 d. n = 3 clones for each clone n = 2 technical replicates. ( D ) Top: mRNA expression levels of mesoderm-specific markers, FOXF1, IRX3, and HAND1 in WT hESCs, control, and FUT1 + ECs on day 0 and after differentiation into LM for 2 d. n = 3 clones for each clone n = 2 technical replicates. Bottom: Representative histograms showing relative counts of WT hESCs and FUT1 + ECs expressing HAND1 protein and quantification of the percent of positive cells for HAND1 in WT hESCs and FUT1 + ECs after LM differentiation for 3 d. n = 3 technical replicates. ( E ) Top: mRNA expression levels of mesoderm-specific markers, NKX2.5, ISL1, and HOPX in WT hESCs, control, and FUT1 + ECs on day 0 and after differentiation into LM for 3 d. n = 3 clones for each clone n = 2 technical replicates. Bottom: Representative histograms showing relative counts of WT hESCs and FUT1 + ECs expressing HOPX protein and quantification of the percent of positive cells for HOPX in WT hESCs and FUT1 + ECs after differentiation into LM for 3 d. Pools of FUT1 + ECs were used for FACS. n = 3 technical replicates. ( F ) Top Left: Schematic showing hESC differentiation into DE over three days by using the protocol of (Loh et al, ). Top right: mRNA expression levels of endoderm-specific markers, HHEX, FOXA2, and SOX17 in WT hESCs, control, and FUT1 + ECs on day 0 and after differentiation into DE for 3 d, as measured by qPCR. n = 2 clones for each clone n = 3 technical replicates. Bottom: Representative histograms showing relative counts of WT hESCs and FUT1 + ECs expressing SOX17 protein and quantification of the fraction of positive cells for SOX17 in WT hESCs and FUT1 + ECs after differentiation into DE for 3 d. n = 3 technical replicates. ( G ) Left: mRNA expression levels of Ectoderm-specific marker, PAX6 in WT hESCs, control, and FUT1 + ECs on day 0 and after differentiation into NPCs for 3 d, as measured by qPCR. n = 2 clones for each clone n = 3 technical replicates. Center: Representative histograms showing relative counts of WT hESCs and FUT1 + ECs expressing PAX6 protein of the fraction of positive cells for PAX6 in WT hESCs and FUT1 + ECs after differentiation into NPCs for 3 d. n = 3 technical replicates. Right: Quantification of positive cells for PAX6 in WT hESCs and FUT1 + ECs after differentiation into NPCs for 3 d. n = 2 clones for each clone n = 2 technical replicates. ( H ) Left: Representative histograms showing relative counts of control and FUT1 + ECs expressing cTnT protein after differentiation into CMs for 20 d. n = 2 clones for each clone n = 3 technical replicates. Left at center: Quantification of the fraction of positive cells for cTnT in control and FUT1 + ECs after differentiation into CMs for 20 d. Left at right: MFI of control and FUT1 + ECs expressing cTnT after differentiation into CMs for 20 d. n = 2 clones for each clone n = 3 technical replicates, Right at left: Representative histograms showing relative counts of control and FUT1 + ECs expressing Myosin protein after differentiation into CMs for 20 d. n = 2 clones for each clone n = 3 technical replicates. Right at center: Quantification of the fraction of positive cells for Myosin in control and FUT1 + ECs after differentiation into CMs for 20 d. Right at right: MFI of control and FUT1 + ECs expressing Myosin after differentiation into CMs for 20 d. n = 2 clones for each clone n = 3 technical replicates. ( I ) Left: Quantification of cardiac marker, TNNT2 mRNA expression in control and FUT1 + ECs on day 0 and after CM differentiation for 20 d, as measured by qPCR. Right: Quantification of cardiac marker, ACTC1 mRNA expression in control and FUT1 + ECs on day 0 and after CM differentiation for 20 d, as measured by qPCR. The housekeeping gene GAPDH, was used for normalization. n = 2 clones for each clone n = 3 technical replicates. More than n = 7 clones of control hESCs and FUT1 + ECs were generated for overexpression experiments; mRNA expression of n = 3 clones was measured by qPCR. Pools and n = 2 clones of control hESCs and FUT1 + ECs originating from WA09-transfected hESCs after sorting were used for imaging and FACS. n = 2 clones of control and FUT1 + ECs were differentiated into CMs and measured by qPCR and FACS. Data presented are relative to the values of day 0 WT hESCs. Data information: In ( A , B ), scale bars represent 100 μm. In ( A – F ), data are presented as means ± SDs. Two-tailed Student’s t-tests * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 or non-significant (NS). In ( G ), data are presented as means ± SDs. Ordinary one-way ANOVA ** P < 0.01 or non-significant (NS), and for PAX 6, Two-tailed Student’s t -test * p < 0.05. In ( H ), data are presented as means ± SDs. Two-tailed Student’s t-tests * p < 0.05, ** p < 0.01, and for Myosin, Ordinary one-way ANOVA ** p < 0.01, **** p < 0.0001. In ( I ), Data are presented as means ± SEM. Two-tailed Student’s t-tests * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: NKX2.5 , Santa Cruz Biotechnology (mouse, monoclonal) (FACS) , , sc-376565.

Techniques: Expressing, Control, Transfection, Clone Assay, Fluorescence, Marker, Generated, Over Expression, Imaging, Two Tailed Test

Journal: EMBO Reports

Article Title: Terminal α1,2-fucosylation of glycosphingolipids by FUT1 is a key regulator in early cell-fate decisions

doi: 10.1038/s44319-024-00243-1

Figure Lengend Snippet: ( A ) Left at left: Representative histograms showing relative counts of WT hESCs expressing α-fucose residues before BMP4 treatment for 48 h. Left at center: Representative histograms showing relative counts of WT hESCs expressing α-fucose residues after BMP4 treatment for 48 h. Left at right: Representative histograms showing relative counts of WT hESCs expressing α-fucose residues after LDN193189 treatment for 48 h. Right at left: MFI of WT hESCs expressing α-fucose residues before and after BMP4 or LDN193189 treatment for 48 h. Right at right: quantification of the percent of positive WT hESCs expressing α-fucose residues before and after BMP4 or LDN193189 treatment for 48 h. n = 6 technical replicates. ( B ) Left at left: Representative histograms showing relative counts of FUT1 + ECs expressing α-fucose residues before and after BMP4 treatment for 48 h. Left at center: Representative histograms showing relative counts of FUT1 + ECs expressing α-fucose residues after BMP4 treatment for 48 h. Left at right: Representative histograms showing relative counts of FUT1 + ECs expressing α-fucose residues before and after LDN193189 treatment for 48 h. Right: MFI and quantification of the percent of positive FUT1 + ECs expressing α-fucose residues before and after BMP4 or LDN193189 treatment for 48 h. n = 6 technical replicates. ( C ) Left at left: Representative histograms showing relative counts of control ECs expressing α-fucose residues before and after BMP4 treatment for 48 h. Left at center: Representative histograms showing relative counts of control ECs expressing α-fucose residues after BMP4 treatment for 48 h. Left at right: Representative histograms showing relative counts of control ECs expressing α-fucose residues before and after LDN193189 treatment for 48 h. Right: MFI and quantification of the percent of positive control ECs expressing α-fucose residues before and after BMP4 or LDN193189 treatments for 48 h. n = 2 clones were analyzed for each experimental condition. n = 3 technical replicates. ( D ) Heatmap comparing the expression level of 50 genes involved in pluripotency and BMP signaling of control ECs and FUT1 + ECs after BMP4 (50 ng/ml) treatment for 48 h. The housekeeping gene GAPDH was used for normalization. n = 3 technical replicates. ( E ) Top at left: Representative histograms showing WT hESCs expressing phosphorylated SMAD1/5/8 after BMP4 (50 ng/ml) treatment for 24 h. Top at right: Quantification of the percent of positive WT hESCs expressing activated SMAD1/5/8 after BMP4 (50 ng/ml) treatment for 24 h. Center at left: Representative histograms showing FUT1 + ECs expressing phosphorylated Smad1/5/8 after BMP4 (50 ng/ml) treatment for 24 h. Center at right: Quantification of the percent of positive control hESCs expressing activated Smad1/5/8 after BMP4 (50 ng/ml) treatment for 24 h. Bottom at left: Representative histograms showing control ECs expressing phosphorylated SMAD1/5/8 after BMP4 (50 ng/ml) treatment for 24 h. Bottom at right: Quantification of the percent of positive control ECs expressing activated Smad1/5/8 after BMP4 (50 ng/ml) treatment for 24 h. n = 2 clones of control and FUT1 + ECs were used for the FACS analysis. n = 3 technical replicates. Data information: In ( A, B, C ), data are presented as means ± SD. Ordinary One-way ANOVA ** p < 0.01, *** p < 0.001, **** p < 0.0001 or non-significant (NS). In ( D ), data are presented as means ± SD. DESeq tests p < 0.05. In ( E ), data are means ± SEM. Two-tailed Student’s t-tests * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: NKX2.5 , Santa Cruz Biotechnology (mouse, monoclonal) (FACS) , , sc-376565.

Techniques: Expressing, Control, Positive Control, Clone Assay, Two Tailed Test

Journal: EMBO Reports

Article Title: Terminal α1,2-fucosylation of glycosphingolipids by FUT1 is a key regulator in early cell-fate decisions

doi: 10.1038/s44319-024-00243-1

Figure Lengend Snippet: Reagents and tools table

Article Snippet: NKX2.5 , Santa Cruz Biotechnology (mouse, monoclonal) (FACS) , , sc-376565.

Techniques: Immunohistochemistry-Frozen IF, Western Blot, Recombinant, Staining, Plasmid Preparation, Sequencing, Control, Transfection, Over Expression, Software, TaqMan Assay, Gene Expression, Binding Assay, Marker

Journal: EMBO Reports

Article Title: Terminal α1,2-fucosylation of glycosphingolipids by FUT1 is a key regulator in early cell-fate decisions

doi: 10.1038/s44319-024-00243-1

Figure Lengend Snippet: Reagents and tools table

Article Snippet: α-Fetoprotein (αFP) , R&D Systems (mouse, monoclonal) (FACS) , , MAB1368.

Techniques: Immunohistochemistry-Frozen IF, Western Blot, Recombinant, Staining, Plasmid Preparation, Sequencing, Control, Transfection, Over Expression, Software, TaqMan Assay, Expressing, Binding Assay, Marker

Journal: eLife

Article Title: Concomitant activation of GLI1 and Notch1 contributes to racial disparity of human triple negative breast cancer progression

doi: 10.7554/eLife.70729

Figure Lengend Snippet: ( A ) ALDH activity assay for dissociated tumor cells from HCC1937 and HCC1806 tumors treated with DAPT + GANT61 and doxorubicin + carboplatin combination. ( B ) Line graph shows tumor progression curves of HCC1806-derived tumors (n = 6) treated with vehicle, doxorubicin, carboplatin, GANT61, DAPT, DAPT + GANT61 + doxorubicin and CARBO + GANT61 + DAPTplatin. ( C ) Representative images of Ki67 stained tumor sections from HCC1806-derived tumors. Scale bar, 100 µm. ( D–H ) Resected tumors from control and treated groups were dissociated and isolated tumor cells were subjected to in vitro functional assays. ( D ) Bar graph shows the migration potential of tumor cells isolated from various treatment groups and control. ( E ) Bar graph shows the number of tumor cells invaded through Matrigel. ( F ) Tumor cell spheroids were formed and allowed to migrate. Representative images of spheroids are shown. Graph shows average distance migrated. Data represents n = 3 independent experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. ( G ) Bar graph shows quantification of solid mammospheres formed from various numbers of tumor cells isolated from various treatment groups and control. Data represents n = 3 independent experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. ( H ) Representative images of mammospheres formed from tumor-dissociated cells. ( I ) Flow cytometry analysis of dissociated tumor cells using CD44 and CD24 staining. ( J ) Representative images of Sox2 and Oct4 stained tumor sections from HCC1806-derived tumors from control and treatment groups. Scale bar, 100 µm.

Article Snippet: Antibody , CD44-PE (FACS)(Mouse monoclonal) , BD Biosciences, San Jose, CA , 550989 , 1:500 dilution.

Techniques: Activity Assay, Derivative Assay, Staining, Isolation, In Vitro, Functional Assay, Migration, Flow Cytometry

Journal: eLife

Article Title: Concomitant activation of GLI1 and Notch1 contributes to racial disparity of human triple negative breast cancer progression

doi: 10.7554/eLife.70729

Figure Lengend Snippet: ( A–B ) Resected secondary tumors from in vivo limiting dilution assay (groups: vehicle, carboplatin (Carbo), or carboplatin + GANT61 + DAPT [CGD]) were dissociated into single cell suspension and subjected to flow cytometry analysis using CD44 and CD49f staining or CD44 and CD24 staining. ( C ) Bar graph shows quantitative representation of CD44 + /CD49 f + and CD44 + /CD24 - cells.

Article Snippet: Antibody , CD44-PE (FACS)(Mouse monoclonal) , BD Biosciences, San Jose, CA , 550989 , 1:500 dilution.

Techniques: In Vivo, Limiting Dilution Assay, Flow Cytometry, Staining

Journal: eLife

Article Title: Concomitant activation of GLI1 and Notch1 contributes to racial disparity of human triple negative breast cancer progression

doi: 10.7554/eLife.70729

Figure Lengend Snippet:

Article Snippet: Antibody , CD44-PE (FACS)(Mouse monoclonal) , BD Biosciences, San Jose, CA , 550989 , 1:500 dilution.

Techniques: In Vitro, In Vivo