mouse gpc5  (OriGene)

Journal: bioRxiv

Article Title: Astrocyte transcriptomic analysis identifies glypican 5 downregulation as a contributor to synaptic dysfunction in Alzheimer’s disease models

doi: 10.1101/2024.10.30.621182

Figure Lengend Snippet: a-b) Correlation between Log2FC of gene expression in 12-month-old APP (a) and Tau (b) mice and human AD DEGs in astrocytes obtained from Grubman et al. Red and blue dots depict up and downregulated genes respectively in APP (a) or Tau mice (b). Grey dots are genes exclusively significant in human astrocytes. Complete gene list can be found in Supplementary Table 12. c) Heatmap showing Log2FC in synapse-related genes in astrocytes from postmortem human AD patients and in 12-month-old APP and Tau mice. Human data obtained from – . * padj <0.05. d) Immunostaining of postmortem human frontal cortex showing GPC5 protein expression in astrocytes marked with GFAP in layer 1. Scale bar = 20 µm. e-f) Representative image showing Gpc5 mRNA in situ hybridization in APP 12-month-old hippocampus (left, scale bar = 100 µm). Right: Zoom in panel showing Gpc5 mRNA levels (white), astrocytes (marked with s100b, magenta) and amyloid plaques (stained with 6e10, green). Scale bar = 20 µm (e). f) Quantification of Gpc5 mRNA signal (% area) in astrocytes associated with amyloid plaques (plaques) or non-associated with amyloid plaques (no plaques). Each datapoint is a mouse (open circles=female, close=male). N = 7 (4F, 3M). Paired t-test was performed for statistical analysis.

Article Snippet: For the overexpression of HA-tagged GPC5, cDNA for the coding sequence of mouse GPC5 (Origene # MR218095) was used.

Techniques: Expressing, Immunostaining, In Situ Hybridization, Staining

Journal: bioRxiv

Article Title: Astrocyte transcriptomic analysis identifies glypican 5 downregulation as a contributor to synaptic dysfunction in Alzheimer’s disease models

doi: 10.1101/2024.10.30.621182

Figure Lengend Snippet: a-b) Diagram depicting a) AAV-PHP.eB expressing HA-Gpc5 or smFP as control under the astrocyte-specific minimal GFAP promoter; b) APP 2-month-old mice were retro-orbitally injected with AAV-HA-Gpc5 or AAV-smFP-HA as control, and at 4 months of age mice were collected for electrophysiology recordings. Same image is shown in . c-d) Representative image of 7-month-old hippocampi from mice injected with either HA-GPC5 or smFP-HA and stained with HA and s100b antibodies. Same images are shown in . Scale bar=20 µm. d) Quantification showing the percentage of s100b-positive astrocytes overexpressing the HA-Gpc5 or smFP-HA construct in the hippocampus. N=3 mice per group, statistical test: T test. e-h) Whole cell patch clamp recordings of spontaneous excitatory postsynaptic currents (sEPSC) in hippocampal CA1 pyramidal neurons. e) Diagram showing the hippocampal neurons recorded in CA1. f) Representative traces from different groups showing an increased frequency of sEPSC in the APP smFP group that is prevented APP GPC5 overexpressing group. g-h) Average frequency (g) and amplitude (h) of sEPSC events during 5-minute recordings. Each data point represents an independent neuron. n= WT smFP: 19 neurons, 9 mice, WT GPC5: 18 neurons, 10 mice, APP smFP: 15 neurons, 10 mice; APP GPC5: 16 neurons, 8 mice. Male (close circles) and female (open circles) mice were included for the analysis. Statistics: 2-way-ANOVA Tukey’s correction for multiple comparison run on neurons. * p <0.05, ** p <0.01, *** p <0.001. Graphs show the mean ± SEM.

Article Snippet: For the overexpression of HA-tagged GPC5, cDNA for the coding sequence of mouse GPC5 (Origene # MR218095) was used.

Techniques: Expressing, Control, Injection, Staining, Construct, Patch Clamp, Comparison

Journal: bioRxiv

Article Title: Astrocyte transcriptomic analysis identifies glypican 5 downregulation as a contributor to synaptic dysfunction in Alzheimer’s disease models

doi: 10.1101/2024.10.30.621182

Figure Lengend Snippet: a) APP and WT 2-month-old mice were retro-orbitally injected with AAV-HA-GPC5 or AAV-smFP as control. At 6 months of age mice were behaviorally tested in the open field and Barnes maze test, at 7 months brains were collected for immunohistochemistry analysis. b) Barnes maze memory test: mice were trained for 5 consecutive days (2 trials a day) to find an escape box. The next 3 days the escape box location is changed to the opposite hole to test cognitive flexibility. On days 6 and 10 a probe trial is performed when the box is removed. c) Representative trajectories used by mice to find the escape box during standard learning (day 2). d) Mean latency to find the target hole is plotted across different training days (mean of the 2 trials per day). Each panel shows different experimental groups. Statistics are calculated using 2-way ANOVA with Dunnett’s multiple comparisons test relative to day 1. WT smFP: N=27(13F, 14M); APP smFP: N=21 (10F, 11M); WT GPC5: N=24 (13F, 11M), APP GPC5: N=18 (7F, 11M). e) Learning slope between day 1 and day 2 (mean latency day 1-mean latency day 2). Statistical analysis is calculated using one-sample T test against hypothetical value of 0 and corrected for multiple comparisons. Each data point represents an independent mouse. (N same as in panel d). f-g) Representative tile scans stitched images of GluA2 immunostaining in the hippocampal CA3 region in 7-month-old APP and WT littermates overexpressing AAV-Gpc5 or smFP. Scale bar = 20 µm (f). Quantification of GluA2 coverage showed a significant increase in GPC5-overexpressing mice (2-way ANOVA treatment effect: * p = 0.02). WT smFP: N=13 (6F, 7M); WT GPC5: N=12 (6F, 6M); APP smFP: N=12 (5F, 7M); APP GPC5: N=11(5F, 6M). Scale bar= 100 µm. h-i) Pearson correlation between GluA2 area and number of smFP (h) or GPC5 (i)-overexpressing astrocytes showed a significant correlation ( p =0.04) only in the GPC5 overexpressing group (i). Statistics: * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001

Article Snippet: For the overexpression of HA-tagged GPC5, cDNA for the coding sequence of mouse GPC5 (Origene # MR218095) was used.

Techniques: Injection, Control, Immunohistochemistry, Immunostaining

Journal: bioRxiv

Article Title: Astrocyte transcriptomic analysis identifies glypican 5 downregulation as a contributor to synaptic dysfunction in Alzheimer’s disease models

doi: 10.1101/2024.10.30.621182

Figure Lengend Snippet: a) Venn diagram showing overlapping upregulated (left panel) and downregulated (right panel) DEGs in 12 month-old APP and Tau mice and human AD astrocytes obtained from Grubman et al. b) Single nuclear RNA sequencing studies from human postmortem patients of different neurodegenerative disorders showing GPC5 downregulation in the astrocyte cluster. Data obtained from – , , . PFC=prefrontal cortex, EC=entorhinal cortex, CTX=cortex, TH=Thalamus, FC=Frontal cortex. c) Synapse-regulating genes are downregulated in GFAP-high astrocyte clusters relative to other clusters in human AD postmortem brains. Data obtained from , , . d) Example image of immunostaining of postmortem human frontal cortex showing GPC5 protein expression in astrocytes marked with GFAP. Scale bar=100 µm. e) Barplot showing TPMs for Gpc5 expression at 4, 6 and 12 months in APP mice. Statistics show the padj value calculated using Deseq2 package in R.

Article Snippet: For the overexpression of HA-tagged GPC5, cDNA for the coding sequence of mouse GPC5 (Origene # MR218095) was used.

Techniques: RNA Sequencing Assay, Immunostaining, Expressing

Journal: bioRxiv

Article Title: Astrocyte transcriptomic analysis identifies glypican 5 downregulation as a contributor to synaptic dysfunction in Alzheimer’s disease models

doi: 10.1101/2024.10.30.621182

Figure Lengend Snippet: Immunohistochemistry analysis of WT and APP 7-month-old mice overexpressing HA-GPC5 or smFP-HA from 2 to 7 months after testing for memory performance in Barnes maze test. a) Left: Sagittal section of a 7-month-old mouse overexpressing HA-GPC5 for 5 months and immunostained with anti-GPC5 antibody showing GPC5 protein being overexpressed throughout the brain. Scale bar=1mm. Right: Magnification showing hippocampal astrocytes overexpressing GPC5 protein, scale bar=100 µm. Both images were stitched image from a tile scan. b-c) Representative hippocampal image (a) and quantification (b) of 7-month-old mouse overexpressing HA-GPC5 or smFP-HA showing colocalization between HA and the astrocyte marker s100b. Scale bar=20 µm. Same image and quantification were shown in main . N= 9-13 mice per group. Male (closed circles) and female mice (open circles) included in the analysis. Statistics: 2-way ANOVA. d-e) Representative hippocampal image showing lack of colocalization between HA and the neuronal marker NeuN in 7-month-old smFP and GPC5-overexpressing mice. e) Quantification showing less than 2% of neurons overexpressing smFP-HA or HA-GPC5. N= 3 mice per group. Statistical analysis: T test.

Article Snippet: For the overexpression of HA-tagged GPC5, cDNA for the coding sequence of mouse GPC5 (Origene # MR218095) was used.

Techniques: Immunohistochemistry, Marker

Journal: bioRxiv

Article Title: Astrocyte transcriptomic analysis identifies glypican 5 downregulation as a contributor to synaptic dysfunction in Alzheimer’s disease models

doi: 10.1101/2024.10.30.621182

Figure Lengend Snippet: a) Representative image (left) and quantification (right) showing colocalization of GFAP-positive astrocytes with HA tag in 4-month-old hippocampus overexpressing smFP-HA or HA-GPC5 for 2 months. Stainings were performed in hippocampal acute slices after electrophysiology recordings were completed. Scale bar=20 µm. Each data point represents an independent mouse. N=3 mice/group. b-c) Representative image (b) and quantification (c) of GFAP staining in the hippocampal CA1 region in WT and APP 4-month-old mice overexpressing HA-GPC5 or smFP control for 2 months. Scale bar=50 µm. Stainings were performed in hippocampal acute slices after electrophysiology recordings were completed. Each data point represents an independent mouse. N=4-6 mice/group. Male (close circles) and female (open circles) mice were included for the analysis. d-f) Electrophysiology recordings of CA1 hippocampal pyramidal neurons performed in 4-month-old APP and WT mice overexpressing HA-GPC5 or smFP showing: d) Resting membrane potential, e) Average decay time and f) average rise time (10-90%) of sEPSC events during 5 minutes recordings. Each data point represents an independent neuron. N= WT smFP: 19 neurons, 9 mice, WT GPC5: 18 neurons, 10 mice, APP smFP: 15 neurons, 10 mice; APP GPC5: 16 neurons, 8 mice. Male (close circles) and female (open circles) mice were included for the analysis. Statistics: 2-way-ANOVA Tukey’s correction for multiple comparison based on neurons. Graphs show the mean ± SEM.

Article Snippet: For the overexpression of HA-tagged GPC5, cDNA for the coding sequence of mouse GPC5 (Origene # MR218095) was used.

Techniques: Staining, Control, Membrane, Comparison

Journal: bioRxiv

Article Title: Astrocyte transcriptomic analysis identifies glypican 5 downregulation as a contributor to synaptic dysfunction in Alzheimer’s disease models

doi: 10.1101/2024.10.30.621182

Figure Lengend Snippet: a-c) Open field test consisting of 10 minutes of spontaneous exploration showed an increased total distance travelled in 6-month-old APP mice compared to WT independently of GPC5 overexpression (a) with no changes in mean speed (b) and time spent in the center of the arena (c). d-e) Barnes maze test: area under the curve for the standard learning curve (d) and time spent exploring the target hole during probe trial (e) revealed no differences between groups. Statistical analysis: 2-way-ANOVA. f-g) Area under the curve for the reversal learning curve in the Bares maze test revealed a significant GPC5 treatment effect (2-way-ANOVA, p =0.03) (f) and no differences in the time spent exploring the target hole during the reversal probe trial (g). h-i) Representative trajectories used by mice to find the escape box during reversal learning (day 2) (h). Mean latency to find the target hole across reversal learning days (mean of the 2 trials per day). Each panel shows different experimental group. Statistics are calculated using 2-way ANOVA with Dunnett’s multiple comparisons test relative to day 1 (i). Statistics: * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Each data point represents an independent mouse. Male (filled dots) and female (open dots) mice were included for the analysis. WT smFP N=27 (13F, 14M); APP smFP N=21 (10F, 11M); WT GPC5 N=24 (13F, 11M), APP GPC5 N=18 (7F, 11M). Graphs show the mean ± SEM.

Article Snippet: For the overexpression of HA-tagged GPC5, cDNA for the coding sequence of mouse GPC5 (Origene # MR218095) was used.

Techniques: Over Expression

Journal: bioRxiv

Article Title: Astrocyte transcriptomic analysis identifies glypican 5 downregulation as a contributor to synaptic dysfunction in Alzheimer’s disease models

doi: 10.1101/2024.10.30.621182

Figure Lengend Snippet: Immunohistochemistry analysis of WT and APP mice overexpressing HA-GPC5 or smFP-HA from 2 to 7 months after testing for memory performance in Barnes maze test. a-c) Representative tile scans stitched image (a) and quantification (b) of hippocampal amyloid plaques stained with the OC antibody in APP 7-month-old mice. Statistics: T-test. smFP N=11 (5F, 6M); GPC5 N=11 (4F, 7M). Scale bar=100 µm. c) Pearson correlation analysis between plaque load and number of HA-positive astrocytes in smFP-overexpression (left panel) or HA-Gpc5 overexpression (right panel) conditions N=10 smFP, 8 GPC5. d-f) Representative tile scans stitched image (d) and quantification (e) of hippocampal GFAP immunoreactivity in the entire hippocampus across different experimental groups. Pearson correlation analysis between GFAP area and number of HA-positive astrocytes in smFP overexpression (left panel) or HA-Gpc5 overexpression (right panel) conditions (f). WT smFP N=13 (6F, 7M); WT GPC5 N=11 (6F, 5M) APP smFP N=11 (5F, 6M); APP GPC5 N=11 (4F, 7M). Scale bar=100 µm. g-i) Representative tile scans stitched images (g) and quantification (h) of hippocampal CA3 vGlut1 immunoreactivity across different experimental groups. Pearson correlation analysis between vGlut1 area and number of HA-positive astrocytes in smFP-overexpression (left panel) or HA-Gpc5 overexpression (right panel) conditions (i). WT smFP N=13 (6F, 7M); WT GPC5 N=12 (6F, 6M); APP smFP N=12 (5F, 7M); APP GPC5 N=12 (5F, 7M). Scale bar=50 µm. j-l) Representative tile scans stitched image (j) and quantification (k) of hippocampal CA3 synaptoporin immunoreactivity across different experimental groups. Pearson correlation analysis between synaptoporin area and number of HA-positive astrocytes in smFP-overexpression (left panel) or HA-Gpc5 overexpression (right panel) conditions (l). WT smFP N=11 (5F, 6M); WT GPC5 N=12 (6F, 6M); APP smFP N=10 (4F, 6M); APP GPC5 N=11 (5F, 6M) Scale bar=50 µm. Graphs show the mean ± SEM. Each data point represents an independent mouse. Male (filled dots) and female (open dots) mice were included for the analysis. Statistics: 2-way ANOVA Tukey’s test for multiple comparisons, unless specified otherwise.

Article Snippet: For the overexpression of HA-tagged GPC5, cDNA for the coding sequence of mouse GPC5 (Origene # MR218095) was used.

Techniques: Immunohistochemistry, Staining, Over Expression

Journal: bioRxiv

Article Title: Astrocyte transcriptomic analysis identifies glypican 5 downregulation as a contributor to synaptic dysfunction in Alzheimer’s disease models

doi: 10.1101/2024.10.30.621182

Figure Lengend Snippet: a-b) Correlation between Log2FC of gene expression in 12-month-old APP (a) and Tau (b) mice and human AD DEGs in astrocytes obtained from Grubman et al. Red and blue dots depict up and downregulated genes respectively in APP (a) or Tau mice (b). Grey dots are genes exclusively significant in human astrocytes. Complete gene list can be found in Supplementary Table 12. c) Heatmap showing Log2FC in synapse-related genes in astrocytes from postmortem human AD patients and in 12-month-old APP and Tau mice. Human data obtained from – . * padj <0.05. d) Immunostaining of postmortem human frontal cortex showing GPC5 protein expression in astrocytes marked with GFAP in layer 1. Scale bar = 20 µm. e-f) Representative image showing Gpc5 mRNA in situ hybridization in APP 12-month-old hippocampus (left, scale bar = 100 µm). Right: Zoom in panel showing Gpc5 mRNA levels (white), astrocytes (marked with s100b, magenta) and amyloid plaques (stained with 6e10, green). Scale bar = 20 µm (e). f) Quantification of Gpc5 mRNA signal (% area) in astrocytes associated with amyloid plaques (plaques) or non-associated with amyloid plaques (no plaques). Each datapoint is a mouse (open circles=female, close=male). N = 7 (4F, 3M). Paired t-test was performed for statistical analysis.

Article Snippet: For the overexpression of HA-tagged GPC5, cDNA for the coding sequence of mouse GPC5 (Origene # MR218095) was used.

Techniques: Expressing, Immunostaining, In Situ Hybridization, Staining

Journal: bioRxiv

Article Title: Astrocyte transcriptomic analysis identifies glypican 5 downregulation as a contributor to synaptic dysfunction in Alzheimer’s disease models

doi: 10.1101/2024.10.30.621182

Figure Lengend Snippet: a-b) Diagram depicting a) AAV-PHP.eB expressing HA-Gpc5 or smFP as control under the astrocyte-specific minimal GFAP promoter; b) APP 2-month-old mice were retro-orbitally injected with AAV-HA-Gpc5 or AAV-smFP-HA as control, and at 4 months of age mice were collected for electrophysiology recordings. Same image is shown in . c-d) Representative image of 7-month-old hippocampi from mice injected with either HA-GPC5 or smFP-HA and stained with HA and s100b antibodies. Same images are shown in . Scale bar=20 µm. d) Quantification showing the percentage of s100b-positive astrocytes overexpressing the HA-Gpc5 or smFP-HA construct in the hippocampus. N=3 mice per group, statistical test: T test. e-h) Whole cell patch clamp recordings of spontaneous excitatory postsynaptic currents (sEPSC) in hippocampal CA1 pyramidal neurons. e) Diagram showing the hippocampal neurons recorded in CA1. f) Representative traces from different groups showing an increased frequency of sEPSC in the APP smFP group that is prevented APP GPC5 overexpressing group. g-h) Average frequency (g) and amplitude (h) of sEPSC events during 5-minute recordings. Each data point represents an independent neuron. n= WT smFP: 19 neurons, 9 mice, WT GPC5: 18 neurons, 10 mice, APP smFP: 15 neurons, 10 mice; APP GPC5: 16 neurons, 8 mice. Male (close circles) and female (open circles) mice were included for the analysis. Statistics: 2-way-ANOVA Tukey’s correction for multiple comparison run on neurons. * p <0.05, ** p <0.01, *** p <0.001. Graphs show the mean ± SEM.

Article Snippet: For the overexpression of HA-tagged GPC5, cDNA for the coding sequence of mouse GPC5 (Origene # MR218095) was used.

Techniques: Expressing, Control, Injection, Staining, Construct, Patch Clamp, Comparison

Journal: bioRxiv

Article Title: Astrocyte transcriptomic analysis identifies glypican 5 downregulation as a contributor to synaptic dysfunction in Alzheimer’s disease models

doi: 10.1101/2024.10.30.621182

Figure Lengend Snippet: a) APP and WT 2-month-old mice were retro-orbitally injected with AAV-HA-GPC5 or AAV-smFP as control. At 6 months of age mice were behaviorally tested in the open field and Barnes maze test, at 7 months brains were collected for immunohistochemistry analysis. b) Barnes maze memory test: mice were trained for 5 consecutive days (2 trials a day) to find an escape box. The next 3 days the escape box location is changed to the opposite hole to test cognitive flexibility. On days 6 and 10 a probe trial is performed when the box is removed. c) Representative trajectories used by mice to find the escape box during standard learning (day 2). d) Mean latency to find the target hole is plotted across different training days (mean of the 2 trials per day). Each panel shows different experimental groups. Statistics are calculated using 2-way ANOVA with Dunnett’s multiple comparisons test relative to day 1. WT smFP: N=27(13F, 14M); APP smFP: N=21 (10F, 11M); WT GPC5: N=24 (13F, 11M), APP GPC5: N=18 (7F, 11M). e) Learning slope between day 1 and day 2 (mean latency day 1-mean latency day 2). Statistical analysis is calculated using one-sample T test against hypothetical value of 0 and corrected for multiple comparisons. Each data point represents an independent mouse. (N same as in panel d). f-g) Representative tile scans stitched images of GluA2 immunostaining in the hippocampal CA3 region in 7-month-old APP and WT littermates overexpressing AAV-Gpc5 or smFP. Scale bar = 20 µm (f). Quantification of GluA2 coverage showed a significant increase in GPC5-overexpressing mice (2-way ANOVA treatment effect: * p = 0.02). WT smFP: N=13 (6F, 7M); WT GPC5: N=12 (6F, 6M); APP smFP: N=12 (5F, 7M); APP GPC5: N=11(5F, 6M). Scale bar= 100 µm. h-i) Pearson correlation between GluA2 area and number of smFP (h) or GPC5 (i)-overexpressing astrocytes showed a significant correlation ( p =0.04) only in the GPC5 overexpressing group (i). Statistics: * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001

Article Snippet: For the overexpression of HA-tagged GPC5, cDNA for the coding sequence of mouse GPC5 (Origene # MR218095) was used.

Techniques: Injection, Control, Immunohistochemistry, Immunostaining

Journal: bioRxiv

Article Title: Astrocyte transcriptomic analysis identifies glypican 5 downregulation as a contributor to synaptic dysfunction in Alzheimer’s disease models

doi: 10.1101/2024.10.30.621182

Figure Lengend Snippet: a) Venn diagram showing overlapping upregulated (left panel) and downregulated (right panel) DEGs in 12 month-old APP and Tau mice and human AD astrocytes obtained from Grubman et al. b) Single nuclear RNA sequencing studies from human postmortem patients of different neurodegenerative disorders showing GPC5 downregulation in the astrocyte cluster. Data obtained from – , , . PFC=prefrontal cortex, EC=entorhinal cortex, CTX=cortex, TH=Thalamus, FC=Frontal cortex. c) Synapse-regulating genes are downregulated in GFAP-high astrocyte clusters relative to other clusters in human AD postmortem brains. Data obtained from , , . d) Example image of immunostaining of postmortem human frontal cortex showing GPC5 protein expression in astrocytes marked with GFAP. Scale bar=100 µm. e) Barplot showing TPMs for Gpc5 expression at 4, 6 and 12 months in APP mice. Statistics show the padj value calculated using Deseq2 package in R.

Article Snippet: For the overexpression of HA-tagged GPC5, cDNA for the coding sequence of mouse GPC5 (Origene # MR218095) was used.

Techniques: RNA Sequencing Assay, Immunostaining, Expressing

Journal: bioRxiv

Article Title: Astrocyte transcriptomic analysis identifies glypican 5 downregulation as a contributor to synaptic dysfunction in Alzheimer’s disease models

doi: 10.1101/2024.10.30.621182

Figure Lengend Snippet: Immunohistochemistry analysis of WT and APP 7-month-old mice overexpressing HA-GPC5 or smFP-HA from 2 to 7 months after testing for memory performance in Barnes maze test. a) Left: Sagittal section of a 7-month-old mouse overexpressing HA-GPC5 for 5 months and immunostained with anti-GPC5 antibody showing GPC5 protein being overexpressed throughout the brain. Scale bar=1mm. Right: Magnification showing hippocampal astrocytes overexpressing GPC5 protein, scale bar=100 µm. Both images were stitched image from a tile scan. b-c) Representative hippocampal image (a) and quantification (b) of 7-month-old mouse overexpressing HA-GPC5 or smFP-HA showing colocalization between HA and the astrocyte marker s100b. Scale bar=20 µm. Same image and quantification were shown in main . N= 9-13 mice per group. Male (closed circles) and female mice (open circles) included in the analysis. Statistics: 2-way ANOVA. d-e) Representative hippocampal image showing lack of colocalization between HA and the neuronal marker NeuN in 7-month-old smFP and GPC5-overexpressing mice. e) Quantification showing less than 2% of neurons overexpressing smFP-HA or HA-GPC5. N= 3 mice per group. Statistical analysis: T test.

Article Snippet: For the overexpression of HA-tagged GPC5, cDNA for the coding sequence of mouse GPC5 (Origene # MR218095) was used.

Techniques: Immunohistochemistry, Marker

Journal: bioRxiv

Article Title: Astrocyte transcriptomic analysis identifies glypican 5 downregulation as a contributor to synaptic dysfunction in Alzheimer’s disease models

doi: 10.1101/2024.10.30.621182

Figure Lengend Snippet: a) Representative image (left) and quantification (right) showing colocalization of GFAP-positive astrocytes with HA tag in 4-month-old hippocampus overexpressing smFP-HA or HA-GPC5 for 2 months. Stainings were performed in hippocampal acute slices after electrophysiology recordings were completed. Scale bar=20 µm. Each data point represents an independent mouse. N=3 mice/group. b-c) Representative image (b) and quantification (c) of GFAP staining in the hippocampal CA1 region in WT and APP 4-month-old mice overexpressing HA-GPC5 or smFP control for 2 months. Scale bar=50 µm. Stainings were performed in hippocampal acute slices after electrophysiology recordings were completed. Each data point represents an independent mouse. N=4-6 mice/group. Male (close circles) and female (open circles) mice were included for the analysis. d-f) Electrophysiology recordings of CA1 hippocampal pyramidal neurons performed in 4-month-old APP and WT mice overexpressing HA-GPC5 or smFP showing: d) Resting membrane potential, e) Average decay time and f) average rise time (10-90%) of sEPSC events during 5 minutes recordings. Each data point represents an independent neuron. N= WT smFP: 19 neurons, 9 mice, WT GPC5: 18 neurons, 10 mice, APP smFP: 15 neurons, 10 mice; APP GPC5: 16 neurons, 8 mice. Male (close circles) and female (open circles) mice were included for the analysis. Statistics: 2-way-ANOVA Tukey’s correction for multiple comparison based on neurons. Graphs show the mean ± SEM.

Article Snippet: For the overexpression of HA-tagged GPC5, cDNA for the coding sequence of mouse GPC5 (Origene # MR218095) was used.

Techniques: Staining, Control, Membrane, Comparison

Journal: bioRxiv

Article Title: Astrocyte transcriptomic analysis identifies glypican 5 downregulation as a contributor to synaptic dysfunction in Alzheimer’s disease models

doi: 10.1101/2024.10.30.621182

Figure Lengend Snippet: a-c) Open field test consisting of 10 minutes of spontaneous exploration showed an increased total distance travelled in 6-month-old APP mice compared to WT independently of GPC5 overexpression (a) with no changes in mean speed (b) and time spent in the center of the arena (c). d-e) Barnes maze test: area under the curve for the standard learning curve (d) and time spent exploring the target hole during probe trial (e) revealed no differences between groups. Statistical analysis: 2-way-ANOVA. f-g) Area under the curve for the reversal learning curve in the Bares maze test revealed a significant GPC5 treatment effect (2-way-ANOVA, p =0.03) (f) and no differences in the time spent exploring the target hole during the reversal probe trial (g). h-i) Representative trajectories used by mice to find the escape box during reversal learning (day 2) (h). Mean latency to find the target hole across reversal learning days (mean of the 2 trials per day). Each panel shows different experimental group. Statistics are calculated using 2-way ANOVA with Dunnett’s multiple comparisons test relative to day 1 (i). Statistics: * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Each data point represents an independent mouse. Male (filled dots) and female (open dots) mice were included for the analysis. WT smFP N=27 (13F, 14M); APP smFP N=21 (10F, 11M); WT GPC5 N=24 (13F, 11M), APP GPC5 N=18 (7F, 11M). Graphs show the mean ± SEM.

Article Snippet: For the overexpression of HA-tagged GPC5, cDNA for the coding sequence of mouse GPC5 (Origene # MR218095) was used.

Techniques: Over Expression

Journal: bioRxiv

Article Title: Astrocyte transcriptomic analysis identifies glypican 5 downregulation as a contributor to synaptic dysfunction in Alzheimer’s disease models

doi: 10.1101/2024.10.30.621182

Figure Lengend Snippet: Immunohistochemistry analysis of WT and APP mice overexpressing HA-GPC5 or smFP-HA from 2 to 7 months after testing for memory performance in Barnes maze test. a-c) Representative tile scans stitched image (a) and quantification (b) of hippocampal amyloid plaques stained with the OC antibody in APP 7-month-old mice. Statistics: T-test. smFP N=11 (5F, 6M); GPC5 N=11 (4F, 7M). Scale bar=100 µm. c) Pearson correlation analysis between plaque load and number of HA-positive astrocytes in smFP-overexpression (left panel) or HA-Gpc5 overexpression (right panel) conditions N=10 smFP, 8 GPC5. d-f) Representative tile scans stitched image (d) and quantification (e) of hippocampal GFAP immunoreactivity in the entire hippocampus across different experimental groups. Pearson correlation analysis between GFAP area and number of HA-positive astrocytes in smFP overexpression (left panel) or HA-Gpc5 overexpression (right panel) conditions (f). WT smFP N=13 (6F, 7M); WT GPC5 N=11 (6F, 5M) APP smFP N=11 (5F, 6M); APP GPC5 N=11 (4F, 7M). Scale bar=100 µm. g-i) Representative tile scans stitched images (g) and quantification (h) of hippocampal CA3 vGlut1 immunoreactivity across different experimental groups. Pearson correlation analysis between vGlut1 area and number of HA-positive astrocytes in smFP-overexpression (left panel) or HA-Gpc5 overexpression (right panel) conditions (i). WT smFP N=13 (6F, 7M); WT GPC5 N=12 (6F, 6M); APP smFP N=12 (5F, 7M); APP GPC5 N=12 (5F, 7M). Scale bar=50 µm. j-l) Representative tile scans stitched image (j) and quantification (k) of hippocampal CA3 synaptoporin immunoreactivity across different experimental groups. Pearson correlation analysis between synaptoporin area and number of HA-positive astrocytes in smFP-overexpression (left panel) or HA-Gpc5 overexpression (right panel) conditions (l). WT smFP N=11 (5F, 6M); WT GPC5 N=12 (6F, 6M); APP smFP N=10 (4F, 6M); APP GPC5 N=11 (5F, 6M) Scale bar=50 µm. Graphs show the mean ± SEM. Each data point represents an independent mouse. Male (filled dots) and female (open dots) mice were included for the analysis. Statistics: 2-way ANOVA Tukey’s test for multiple comparisons, unless specified otherwise.

Article Snippet: For the overexpression of HA-tagged GPC5, cDNA for the coding sequence of mouse GPC5 (Origene # MR218095) was used.

Techniques: Immunohistochemistry, Staining, Over Expression

Journal: PLoS ONE

Article Title: Subcellular localization of glypican-5 is associated with dynamic motility of the human mesenchymal stem cell line U3DT

doi: 10.1371/journal.pone.0226538

Figure Lengend Snippet: Gene expression of GPCs (GPC1 to GPC6) at four culture stages were analyzed using data in the DDBJ database of the National Institute of Genetics ( http://www.ddbj.nig.ac.jp/ ), accession number: DRA000533 . Each value is shown relative to the corresponding value in U3-A cells (culture stage 1). GPC1, red; GPC2, green; GPC3, no expression; GPC4, purple; GPC5, blue; GPC6, light blue. As shown in the previous report , PDLs of U3-A, U3-B, U3-C and U3DT cells are 60‒90, 91‒150, 151‒230 and 231‒295, respectively.

Article Snippet: The following antibodies and fluorescence reagents were used: anti-GPC5 antibody (MAB2607, R&D Systems, Inc.), Alexa Fluor 594 conjugated anti-GPC5 antibody (R&D Systems, Inc.), Alexa Fluor 647 conjugated anti-GPC5 antibody (R&D Systems, Inc.), anti-FGFR1 Xp rabbit monoclonal antibody (D8E4, Cell Signaling Technology), anti-Rab11A antibody (A-6: sc-166912, Santa Cruz Biotech), anti-Rab11 (D4F5)XP rabbit monoclonal antibody (Cell Signaling Technology), anti-acetyl-alpha-tubulin rabbit monoclonal antibody (D20G3, #5335, Cell Signaling Technology), Alexa Fluor 488-conjucated wheat germ agglutinin (WGA; W1126: Invitrogen), anti-CD63 monoclonal antibody (MX-49.129.5: sc-5275, Santa Cruz Biotechnology), anti-CD63 monoclonal antibody (cl 3–13: Fuji Film Co.), anti-ARF6 monoclonal antibody (3A-1: sc-7971, Santa Cruz Biotechnology), anti-ARF6 polyclonal rabbit antibody (20225-1-AP, Proteintech), Alexa Fluor 488-conjugated goat-anti-mouse IgG(H+L), F(ab’)2 fragment (#4408, Cell Signaling), Alexa Fluor 488-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4412, Cell Signaling Technology), Alexa Fluor 568-labeled donkey-anti-mouse IgG (H+L) (A10037, Invitrogen), Alexa Fluor 594-labeled goat anti-mouse IgG IgG(H+L), F(ab’)2 fragment (A-11020, Molecular Probes, Inc.), and Alexa Fluor 647-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4414, Cell Signaling Technology).

Techniques: Gene Expression, Expressing

Journal: PLoS ONE

Article Title: Subcellular localization of glypican-5 is associated with dynamic motility of the human mesenchymal stem cell line U3DT

doi: 10.1371/journal.pone.0226538

Figure Lengend Snippet: Double immunofluorescence labeling of U3DT cells with anti-GPC5 (red) and anti-FGFR (green) (top three lines), with anti-GPC5 (red) and anti-ARF (green) (fourth line), or with anti-GPC5 (red) and anti-Ra11A (green) (bottom line) antibodies. Positive spots (yellow) in each merged image were clearly visible in U3DT cells. Scale bar, 5 μm.

Article Snippet: The following antibodies and fluorescence reagents were used: anti-GPC5 antibody (MAB2607, R&D Systems, Inc.), Alexa Fluor 594 conjugated anti-GPC5 antibody (R&D Systems, Inc.), Alexa Fluor 647 conjugated anti-GPC5 antibody (R&D Systems, Inc.), anti-FGFR1 Xp rabbit monoclonal antibody (D8E4, Cell Signaling Technology), anti-Rab11A antibody (A-6: sc-166912, Santa Cruz Biotech), anti-Rab11 (D4F5)XP rabbit monoclonal antibody (Cell Signaling Technology), anti-acetyl-alpha-tubulin rabbit monoclonal antibody (D20G3, #5335, Cell Signaling Technology), Alexa Fluor 488-conjucated wheat germ agglutinin (WGA; W1126: Invitrogen), anti-CD63 monoclonal antibody (MX-49.129.5: sc-5275, Santa Cruz Biotechnology), anti-CD63 monoclonal antibody (cl 3–13: Fuji Film Co.), anti-ARF6 monoclonal antibody (3A-1: sc-7971, Santa Cruz Biotechnology), anti-ARF6 polyclonal rabbit antibody (20225-1-AP, Proteintech), Alexa Fluor 488-conjugated goat-anti-mouse IgG(H+L), F(ab’)2 fragment (#4408, Cell Signaling), Alexa Fluor 488-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4412, Cell Signaling Technology), Alexa Fluor 568-labeled donkey-anti-mouse IgG (H+L) (A10037, Invitrogen), Alexa Fluor 594-labeled goat anti-mouse IgG IgG(H+L), F(ab’)2 fragment (A-11020, Molecular Probes, Inc.), and Alexa Fluor 647-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4414, Cell Signaling Technology).

Techniques: Immunofluorescence, Labeling

Journal: PLoS ONE

Article Title: Subcellular localization of glypican-5 is associated with dynamic motility of the human mesenchymal stem cell line U3DT

doi: 10.1371/journal.pone.0226538

Figure Lengend Snippet: U3DT cells at different stages of mitosis and cytokinesis were fixed but not permeabilized, stained with anti-GPC5 (red) and anti-FGFR1 (green) antibodies, and counterstained with DAPI stain (blue). Yellow represents the degree of colocalization. Scale bar, 5 μm.

Article Snippet: The following antibodies and fluorescence reagents were used: anti-GPC5 antibody (MAB2607, R&D Systems, Inc.), Alexa Fluor 594 conjugated anti-GPC5 antibody (R&D Systems, Inc.), Alexa Fluor 647 conjugated anti-GPC5 antibody (R&D Systems, Inc.), anti-FGFR1 Xp rabbit monoclonal antibody (D8E4, Cell Signaling Technology), anti-Rab11A antibody (A-6: sc-166912, Santa Cruz Biotech), anti-Rab11 (D4F5)XP rabbit monoclonal antibody (Cell Signaling Technology), anti-acetyl-alpha-tubulin rabbit monoclonal antibody (D20G3, #5335, Cell Signaling Technology), Alexa Fluor 488-conjucated wheat germ agglutinin (WGA; W1126: Invitrogen), anti-CD63 monoclonal antibody (MX-49.129.5: sc-5275, Santa Cruz Biotechnology), anti-CD63 monoclonal antibody (cl 3–13: Fuji Film Co.), anti-ARF6 monoclonal antibody (3A-1: sc-7971, Santa Cruz Biotechnology), anti-ARF6 polyclonal rabbit antibody (20225-1-AP, Proteintech), Alexa Fluor 488-conjugated goat-anti-mouse IgG(H+L), F(ab’)2 fragment (#4408, Cell Signaling), Alexa Fluor 488-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4412, Cell Signaling Technology), Alexa Fluor 568-labeled donkey-anti-mouse IgG (H+L) (A10037, Invitrogen), Alexa Fluor 594-labeled goat anti-mouse IgG IgG(H+L), F(ab’)2 fragment (A-11020, Molecular Probes, Inc.), and Alexa Fluor 647-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4414, Cell Signaling Technology).

Techniques: Staining

Journal: PLoS ONE

Article Title: Subcellular localization of glypican-5 is associated with dynamic motility of the human mesenchymal stem cell line U3DT

doi: 10.1371/journal.pone.0226538

Figure Lengend Snippet: (A) Rab11 localized on both sides (red) of the midbody dark zone and partially overlapped with microtubules (gray). Localizations of proteins on midbody microtubules were determined and compared by line scans. Microtubules and Rab11 peaked at the same positions, where the microtubule signal was high and the FGFR signal (green) was low. (B) Rab11 localization (red) adjacent to the midbody. Plasma membrane was stained with WGA (green). (C) GPC5 (red) colocalized with Rab11 (gray) at the midbody. The plasma membrane was stained with WGA (green). (D) GPC5 (red) colocalized with Rab11 (green) on midbody microtubules (gray). Scale bar, 2 μm.

Article Snippet: The following antibodies and fluorescence reagents were used: anti-GPC5 antibody (MAB2607, R&D Systems, Inc.), Alexa Fluor 594 conjugated anti-GPC5 antibody (R&D Systems, Inc.), Alexa Fluor 647 conjugated anti-GPC5 antibody (R&D Systems, Inc.), anti-FGFR1 Xp rabbit monoclonal antibody (D8E4, Cell Signaling Technology), anti-Rab11A antibody (A-6: sc-166912, Santa Cruz Biotech), anti-Rab11 (D4F5)XP rabbit monoclonal antibody (Cell Signaling Technology), anti-acetyl-alpha-tubulin rabbit monoclonal antibody (D20G3, #5335, Cell Signaling Technology), Alexa Fluor 488-conjucated wheat germ agglutinin (WGA; W1126: Invitrogen), anti-CD63 monoclonal antibody (MX-49.129.5: sc-5275, Santa Cruz Biotechnology), anti-CD63 monoclonal antibody (cl 3–13: Fuji Film Co.), anti-ARF6 monoclonal antibody (3A-1: sc-7971, Santa Cruz Biotechnology), anti-ARF6 polyclonal rabbit antibody (20225-1-AP, Proteintech), Alexa Fluor 488-conjugated goat-anti-mouse IgG(H+L), F(ab’)2 fragment (#4408, Cell Signaling), Alexa Fluor 488-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4412, Cell Signaling Technology), Alexa Fluor 568-labeled donkey-anti-mouse IgG (H+L) (A10037, Invitrogen), Alexa Fluor 594-labeled goat anti-mouse IgG IgG(H+L), F(ab’)2 fragment (A-11020, Molecular Probes, Inc.), and Alexa Fluor 647-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4414, Cell Signaling Technology).

Techniques: Clinical Proteomics, Membrane, Staining

Journal: PLoS ONE

Article Title: Subcellular localization of glypican-5 is associated with dynamic motility of the human mesenchymal stem cell line U3DT

doi: 10.1371/journal.pone.0226538

Figure Lengend Snippet: (A‒D) Images of staining with Alexa Fluor 488-conjugated WGA (green) and DAPI (blue) at interphase (A), metaphase (B), and anaphase and telophase (C, D). (D) Maximum projection of 15 Z-staged-images stained with Alexa Fluor 488-WGA and DAPI. (E) U3DT cell at telophase, stained with anti-GPC5 antibody (red), Alexa Fluor 488-WGA (green), and DAPI (blue). (F) Blebs of U3DT cell at telophase stained with anti-FGFR1 (brown), Alexa Fluor 488-WGA (green), and DAPI (blue). (G) Blebs of U3DT cells at telophase stained with anti-Rab11 rabbit antibodies (magenta), Alexa Fluor 488-WGA (green), and DAPI (blue). Scale bar, 5 μm.

Article Snippet: The following antibodies and fluorescence reagents were used: anti-GPC5 antibody (MAB2607, R&D Systems, Inc.), Alexa Fluor 594 conjugated anti-GPC5 antibody (R&D Systems, Inc.), Alexa Fluor 647 conjugated anti-GPC5 antibody (R&D Systems, Inc.), anti-FGFR1 Xp rabbit monoclonal antibody (D8E4, Cell Signaling Technology), anti-Rab11A antibody (A-6: sc-166912, Santa Cruz Biotech), anti-Rab11 (D4F5)XP rabbit monoclonal antibody (Cell Signaling Technology), anti-acetyl-alpha-tubulin rabbit monoclonal antibody (D20G3, #5335, Cell Signaling Technology), Alexa Fluor 488-conjucated wheat germ agglutinin (WGA; W1126: Invitrogen), anti-CD63 monoclonal antibody (MX-49.129.5: sc-5275, Santa Cruz Biotechnology), anti-CD63 monoclonal antibody (cl 3–13: Fuji Film Co.), anti-ARF6 monoclonal antibody (3A-1: sc-7971, Santa Cruz Biotechnology), anti-ARF6 polyclonal rabbit antibody (20225-1-AP, Proteintech), Alexa Fluor 488-conjugated goat-anti-mouse IgG(H+L), F(ab’)2 fragment (#4408, Cell Signaling), Alexa Fluor 488-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4412, Cell Signaling Technology), Alexa Fluor 568-labeled donkey-anti-mouse IgG (H+L) (A10037, Invitrogen), Alexa Fluor 594-labeled goat anti-mouse IgG IgG(H+L), F(ab’)2 fragment (A-11020, Molecular Probes, Inc.), and Alexa Fluor 647-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4414, Cell Signaling Technology).

Techniques: Staining

Journal: PLoS ONE

Article Title: Subcellular localization of glypican-5 is associated with dynamic motility of the human mesenchymal stem cell line U3DT

doi: 10.1371/journal.pone.0226538

Figure Lengend Snippet: Immunofluorescence images of cells were obtained using a Leica SP-8 confocal microscope, and the pixel sum was estimated with the SP8 software (RAS X). (A,B) Images of Rab11 in untreated (A) and RAB11A-siRNA-treated U3DT cells (B). (C) Quantitative analysis of U3DT cells not treated (blue) or treated (red) with 100 nM RAB11A-siRNA. (D,E) Images of GPC5 in U3DT cells not treated (D) or treated (E) with 100 nM RAB11A-siRNA. (F) Quantitative expression of GPC5 in U3DT cells not treated (blue) or treated (red) with 100 nM RAB11A-siRNA. (G,H) Images of GPC5 (red) and Rab11 (green) in trypsinized-U3DT cells not treated (G) or treated (H) with RAB11A-siRNA. (G‒J) Immunofluorescence images (G,H) and the pixel sum (I,J) of trypsinized cells were obtained using a Leica SP-8 immunofluorescence microscope the same as in C and F. (I) The mean intensity (pixel sum) of Rab11 relative to that of DAPI is shown for each untreated (blue) and RAB11A-siRNA-treated U3DT cell (red). (J) The mean fluorescence intensity (pixel sum) of GPC5 relative to that of DAPI staining per cell is shown for each untreated (blue) or RAB11A-siRNA-treated U3DT cell (red). (K‒M) FACS analysis. (K) Contour display of merged three images. U3DT cells were stained with (blue) or without (red) anti-GPC5 and anti-Rab11 antibodies. RAB11A-siRNA-treated cells were stained with anti-GPC5 and anti-Rab11 antibodies (green). (L) Histogram of Alexa Fluor 488 fluorescence intensity (Rab11) of RAB11A-siRNA-treated cells (green) or not treated cells (blue). (M) Histogram of Alexa Fluor 594 fluorescence intensity (GPC5) of RAB11A-siRNA-treated cells (green) or not treated cells (blue). The same preparation was used for immunofluorescence (G, H) and for FACS analyzes (L, M). Red arrows indicate the peak position of immunostaining control cells (L, M). (N, O) Immunofluorescence images of Rab11 (red) and WGA (green) in not treated cells (N) and RAB11A-siRNA-treated cells (O). (P) The mean distribution of telophase cells with three or more blebs (>ca. 2 μm diameter) in telophase are shown for not treated cells (blue) and RAB11A-siRNA-treated cells (red). The numbers in parentheses in Figure (C, F, I, J and P) are the number of images. White arrows indicate blebs in a telophase cell (N). Scale bar, 5 μm.

Article Snippet: The following antibodies and fluorescence reagents were used: anti-GPC5 antibody (MAB2607, R&D Systems, Inc.), Alexa Fluor 594 conjugated anti-GPC5 antibody (R&D Systems, Inc.), Alexa Fluor 647 conjugated anti-GPC5 antibody (R&D Systems, Inc.), anti-FGFR1 Xp rabbit monoclonal antibody (D8E4, Cell Signaling Technology), anti-Rab11A antibody (A-6: sc-166912, Santa Cruz Biotech), anti-Rab11 (D4F5)XP rabbit monoclonal antibody (Cell Signaling Technology), anti-acetyl-alpha-tubulin rabbit monoclonal antibody (D20G3, #5335, Cell Signaling Technology), Alexa Fluor 488-conjucated wheat germ agglutinin (WGA; W1126: Invitrogen), anti-CD63 monoclonal antibody (MX-49.129.5: sc-5275, Santa Cruz Biotechnology), anti-CD63 monoclonal antibody (cl 3–13: Fuji Film Co.), anti-ARF6 monoclonal antibody (3A-1: sc-7971, Santa Cruz Biotechnology), anti-ARF6 polyclonal rabbit antibody (20225-1-AP, Proteintech), Alexa Fluor 488-conjugated goat-anti-mouse IgG(H+L), F(ab’)2 fragment (#4408, Cell Signaling), Alexa Fluor 488-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4412, Cell Signaling Technology), Alexa Fluor 568-labeled donkey-anti-mouse IgG (H+L) (A10037, Invitrogen), Alexa Fluor 594-labeled goat anti-mouse IgG IgG(H+L), F(ab’)2 fragment (A-11020, Molecular Probes, Inc.), and Alexa Fluor 647-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4414, Cell Signaling Technology).

Techniques: Immunofluorescence, Microscopy, Software, Expressing, Fluorescence, Staining, Immunostaining, Control

Journal: PLoS ONE

Article Title: Subcellular localization of glypican-5 is associated with dynamic motility of the human mesenchymal stem cell line U3DT

doi: 10.1371/journal.pone.0226538

Figure Lengend Snippet: (A) Electron microscopic image of negative-stained EVs. The inset shows a magnification of the boxed EV. (B) Size distribution of vesicles in EV preparations measured by image software (n = 110). (C) (F) (I) (L) GPC5-immunostained EVs (red) stained for Rab11 (green) (C), FGFR1 (green) (F), CD63 (green) (I), and ARF6 (green) (L). In (I) and (L), an Alexa Fluor 647- conjugated anti-GPC5 antibody was used to detect of GPC5-ositive particles. (D) (G) (J) (M) Line scan determination of the red bars in (C), (F), (I), and (L): GPC5 (red), others (green). (E) (H) (K) (N) Distribution of GPC5-positive particles identified by scan determinations in (D), (G), (J), and (M). n = 1,516, (E), 630 (H), 573 (K), and 835 (N). Scale bars: (A), 500 nm; (C), (F), (I), (L), 2 μm.

Article Snippet: The following antibodies and fluorescence reagents were used: anti-GPC5 antibody (MAB2607, R&D Systems, Inc.), Alexa Fluor 594 conjugated anti-GPC5 antibody (R&D Systems, Inc.), Alexa Fluor 647 conjugated anti-GPC5 antibody (R&D Systems, Inc.), anti-FGFR1 Xp rabbit monoclonal antibody (D8E4, Cell Signaling Technology), anti-Rab11A antibody (A-6: sc-166912, Santa Cruz Biotech), anti-Rab11 (D4F5)XP rabbit monoclonal antibody (Cell Signaling Technology), anti-acetyl-alpha-tubulin rabbit monoclonal antibody (D20G3, #5335, Cell Signaling Technology), Alexa Fluor 488-conjucated wheat germ agglutinin (WGA; W1126: Invitrogen), anti-CD63 monoclonal antibody (MX-49.129.5: sc-5275, Santa Cruz Biotechnology), anti-CD63 monoclonal antibody (cl 3–13: Fuji Film Co.), anti-ARF6 monoclonal antibody (3A-1: sc-7971, Santa Cruz Biotechnology), anti-ARF6 polyclonal rabbit antibody (20225-1-AP, Proteintech), Alexa Fluor 488-conjugated goat-anti-mouse IgG(H+L), F(ab’)2 fragment (#4408, Cell Signaling), Alexa Fluor 488-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4412, Cell Signaling Technology), Alexa Fluor 568-labeled donkey-anti-mouse IgG (H+L) (A10037, Invitrogen), Alexa Fluor 594-labeled goat anti-mouse IgG IgG(H+L), F(ab’)2 fragment (A-11020, Molecular Probes, Inc.), and Alexa Fluor 647-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4414, Cell Signaling Technology).

Techniques: Staining, Software

Journal: PLoS ONE

Article Title: Subcellular localization of glypican-5 is associated with dynamic motility of the human mesenchymal stem cell line U3DT

doi: 10.1371/journal.pone.0226538

Figure Lengend Snippet: (A, D) Images of control cells. (B, E) Images of EV-treated cells. (D, E) Merged images of UE6E7T-3 cells (D) and EV-treated cells (E) were stained for GPC5 (red) and FGFR1 (green). (C) Quantitation of GPC5 (pixel sum per cell) in control UE6E7T-3 cells (blue) or cells cultured with EVs for 1 day (red). n = 67 (blue) and 78 (red). (F, G) FACS pattern of GPC5 in UE6E7T-3 cells (F) and in cells cultured with EVs for 1 day (G). ⇔ indicates GPC5-positive cells. Scale bar, 5 μm.

Article Snippet: The following antibodies and fluorescence reagents were used: anti-GPC5 antibody (MAB2607, R&D Systems, Inc.), Alexa Fluor 594 conjugated anti-GPC5 antibody (R&D Systems, Inc.), Alexa Fluor 647 conjugated anti-GPC5 antibody (R&D Systems, Inc.), anti-FGFR1 Xp rabbit monoclonal antibody (D8E4, Cell Signaling Technology), anti-Rab11A antibody (A-6: sc-166912, Santa Cruz Biotech), anti-Rab11 (D4F5)XP rabbit monoclonal antibody (Cell Signaling Technology), anti-acetyl-alpha-tubulin rabbit monoclonal antibody (D20G3, #5335, Cell Signaling Technology), Alexa Fluor 488-conjucated wheat germ agglutinin (WGA; W1126: Invitrogen), anti-CD63 monoclonal antibody (MX-49.129.5: sc-5275, Santa Cruz Biotechnology), anti-CD63 monoclonal antibody (cl 3–13: Fuji Film Co.), anti-ARF6 monoclonal antibody (3A-1: sc-7971, Santa Cruz Biotechnology), anti-ARF6 polyclonal rabbit antibody (20225-1-AP, Proteintech), Alexa Fluor 488-conjugated goat-anti-mouse IgG(H+L), F(ab’)2 fragment (#4408, Cell Signaling), Alexa Fluor 488-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4412, Cell Signaling Technology), Alexa Fluor 568-labeled donkey-anti-mouse IgG (H+L) (A10037, Invitrogen), Alexa Fluor 594-labeled goat anti-mouse IgG IgG(H+L), F(ab’)2 fragment (A-11020, Molecular Probes, Inc.), and Alexa Fluor 647-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4414, Cell Signaling Technology).

Techniques: Control, Staining, Quantitation Assay, Cell Culture

Journal: PLoS ONE

Article Title: Subcellular localization of glypican-5 is associated with dynamic motility of the human mesenchymal stem cell line U3DT

doi: 10.1371/journal.pone.0226538

Figure Lengend Snippet: (A) Control cells fixed immediately after scratching. The wide of the scratched area was ca. 500μm. (B‒D) Mock (B), non-targeting siRNA (NT)-treated (C), and GPC5-siRNA (KD)-treated (D) cells were cultured in appropriate medium containing 25 nM FGF2 for 23 h after scratching. Cells were fixed and immunofluorescence images were acquired using a Leica SP-8 microscope equipped with a 20x objective. (E) The number of cells that moved into the scratched area or the removed insert area was counted after incubation of control (without siRNA) cells (yellow), NT cells (red), and KD cells (blue) in appropriate medium containing (red and blue columns) or lacking (brown and sky blue columns) FGF2 at 37°C for 23 h. (F‒H) Immunofluorescence images of cells treated with mock (F), non-targeting siRNA (G), or GPC5-targeting siRNA (H) for 72 h after removing an insert from a μ -Dish. Cells were stained with anti-GPC5 (red) and anti-FGFR1 (green) antibodies. (I) Quantification of the percentage of cells with blebs at telophase. Control (mock) cells (yellow), NT cells (red) and KD cells (blue) were incubated in appropriate medium containing FGF2 at 37°C for 23 h. (J‒L) Immunofluorescence images of mock (J), non-targeting siRNA-treated (K), and GPC5-siRNA-treated (L) cells after 72 h. Cells were stained with WGA (green). White arrows indicate blebs in a telophase cell (J, K). The numbers in parentheses in (E, I) are the number of images observed. Scale bar: (F‒H), 100 μm; (J‒L), 2 μm.

Article Snippet: The following antibodies and fluorescence reagents were used: anti-GPC5 antibody (MAB2607, R&D Systems, Inc.), Alexa Fluor 594 conjugated anti-GPC5 antibody (R&D Systems, Inc.), Alexa Fluor 647 conjugated anti-GPC5 antibody (R&D Systems, Inc.), anti-FGFR1 Xp rabbit monoclonal antibody (D8E4, Cell Signaling Technology), anti-Rab11A antibody (A-6: sc-166912, Santa Cruz Biotech), anti-Rab11 (D4F5)XP rabbit monoclonal antibody (Cell Signaling Technology), anti-acetyl-alpha-tubulin rabbit monoclonal antibody (D20G3, #5335, Cell Signaling Technology), Alexa Fluor 488-conjucated wheat germ agglutinin (WGA; W1126: Invitrogen), anti-CD63 monoclonal antibody (MX-49.129.5: sc-5275, Santa Cruz Biotechnology), anti-CD63 monoclonal antibody (cl 3–13: Fuji Film Co.), anti-ARF6 monoclonal antibody (3A-1: sc-7971, Santa Cruz Biotechnology), anti-ARF6 polyclonal rabbit antibody (20225-1-AP, Proteintech), Alexa Fluor 488-conjugated goat-anti-mouse IgG(H+L), F(ab’)2 fragment (#4408, Cell Signaling), Alexa Fluor 488-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4412, Cell Signaling Technology), Alexa Fluor 568-labeled donkey-anti-mouse IgG (H+L) (A10037, Invitrogen), Alexa Fluor 594-labeled goat anti-mouse IgG IgG(H+L), F(ab’)2 fragment (A-11020, Molecular Probes, Inc.), and Alexa Fluor 647-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4414, Cell Signaling Technology).

Techniques: Control, Cell Culture, Immunofluorescence, Microscopy, Incubation, Staining

Journal: PLoS ONE

Article Title: Subcellular localization of glypican-5 is associated with dynamic motility of the human mesenchymal stem cell line U3DT

doi: 10.1371/journal.pone.0226538

Figure Lengend Snippet: (a) GPC5 (red) localizes at the leading edge of migrating cells, (b) at the equatorial plane, (c) at the furrow, (d) at the intercellular bridge during mitosis, (e) in membrane blebs during cytokinesis, (f) at the tips of filopodia, and (g) in EVs.

Article Snippet: The following antibodies and fluorescence reagents were used: anti-GPC5 antibody (MAB2607, R&D Systems, Inc.), Alexa Fluor 594 conjugated anti-GPC5 antibody (R&D Systems, Inc.), Alexa Fluor 647 conjugated anti-GPC5 antibody (R&D Systems, Inc.), anti-FGFR1 Xp rabbit monoclonal antibody (D8E4, Cell Signaling Technology), anti-Rab11A antibody (A-6: sc-166912, Santa Cruz Biotech), anti-Rab11 (D4F5)XP rabbit monoclonal antibody (Cell Signaling Technology), anti-acetyl-alpha-tubulin rabbit monoclonal antibody (D20G3, #5335, Cell Signaling Technology), Alexa Fluor 488-conjucated wheat germ agglutinin (WGA; W1126: Invitrogen), anti-CD63 monoclonal antibody (MX-49.129.5: sc-5275, Santa Cruz Biotechnology), anti-CD63 monoclonal antibody (cl 3–13: Fuji Film Co.), anti-ARF6 monoclonal antibody (3A-1: sc-7971, Santa Cruz Biotechnology), anti-ARF6 polyclonal rabbit antibody (20225-1-AP, Proteintech), Alexa Fluor 488-conjugated goat-anti-mouse IgG(H+L), F(ab’)2 fragment (#4408, Cell Signaling), Alexa Fluor 488-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4412, Cell Signaling Technology), Alexa Fluor 568-labeled donkey-anti-mouse IgG (H+L) (A10037, Invitrogen), Alexa Fluor 594-labeled goat anti-mouse IgG IgG(H+L), F(ab’)2 fragment (A-11020, Molecular Probes, Inc.), and Alexa Fluor 647-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4414, Cell Signaling Technology).

Techniques: Membrane

Journal: PLoS ONE

Article Title: Subcellular localization of glypican-5 is associated with dynamic motility of the human mesenchymal stem cell line U3DT

doi: 10.1371/journal.pone.0226538

Figure Lengend Snippet: Gene expression of GPCs (GPC1 to GPC6) at four culture stages were analyzed using data in the DDBJ database of the National Institute of Genetics ( http://www.ddbj.nig.ac.jp/ ), accession number: DRA000533 . Each value is shown relative to the corresponding value in U3-A cells (culture stage 1). GPC1, red; GPC2, green; GPC3, no expression; GPC4, purple; GPC5, blue; GPC6, light blue. As shown in the previous report , PDLs of U3-A, U3-B, U3-C and U3DT cells are 60‒90, 91‒150, 151‒230 and 231‒295, respectively.

Article Snippet: The following antibodies and fluorescence reagents were used: anti-GPC5 antibody (MAB2607, R&D Systems, Inc.), Alexa Fluor 594 conjugated anti-GPC5 antibody (R&D Systems, Inc.), Alexa Fluor 647 conjugated anti-GPC5 antibody (R&D Systems, Inc.), anti-FGFR1 Xp rabbit monoclonal antibody (D8E4, Cell Signaling Technology), anti-Rab11A antibody (A-6: sc-166912, Santa Cruz Biotech), anti-Rab11 (D4F5)XP rabbit monoclonal antibody (Cell Signaling Technology), anti-acetyl-alpha-tubulin rabbit monoclonal antibody (D20G3, #5335, Cell Signaling Technology), Alexa Fluor 488-conjucated wheat germ agglutinin (WGA; W1126: Invitrogen), anti-CD63 monoclonal antibody (MX-49.129.5: sc-5275, Santa Cruz Biotechnology), anti-CD63 monoclonal antibody (cl 3–13: Fuji Film Co.), anti-ARF6 monoclonal antibody (3A-1: sc-7971, Santa Cruz Biotechnology), anti-ARF6 polyclonal rabbit antibody (20225-1-AP, Proteintech), Alexa Fluor 488-conjugated goat-anti-mouse IgG(H+L), F(ab’)2 fragment (#4408, Cell Signaling), Alexa Fluor 488-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4412, Cell Signaling Technology), Alexa Fluor 568-labeled donkey-anti-mouse IgG (H+L) (A10037, Invitrogen), Alexa Fluor 594-labeled goat anti-mouse IgG IgG(H+L), F(ab’)2 fragment (A-11020, Molecular Probes, Inc.), and Alexa Fluor 647-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4414, Cell Signaling Technology).

Techniques: Gene Expression, Expressing

Journal: PLoS ONE

Article Title: Subcellular localization of glypican-5 is associated with dynamic motility of the human mesenchymal stem cell line U3DT

doi: 10.1371/journal.pone.0226538

Figure Lengend Snippet: Double immunofluorescence labeling of U3DT cells with anti-GPC5 (red) and anti-FGFR (green) (top three lines), with anti-GPC5 (red) and anti-ARF (green) (fourth line), or with anti-GPC5 (red) and anti-Ra11A (green) (bottom line) antibodies. Positive spots (yellow) in each merged image were clearly visible in U3DT cells. Scale bar, 5 μm.

Article Snippet: The following antibodies and fluorescence reagents were used: anti-GPC5 antibody (MAB2607, R&D Systems, Inc.), Alexa Fluor 594 conjugated anti-GPC5 antibody (R&D Systems, Inc.), Alexa Fluor 647 conjugated anti-GPC5 antibody (R&D Systems, Inc.), anti-FGFR1 Xp rabbit monoclonal antibody (D8E4, Cell Signaling Technology), anti-Rab11A antibody (A-6: sc-166912, Santa Cruz Biotech), anti-Rab11 (D4F5)XP rabbit monoclonal antibody (Cell Signaling Technology), anti-acetyl-alpha-tubulin rabbit monoclonal antibody (D20G3, #5335, Cell Signaling Technology), Alexa Fluor 488-conjucated wheat germ agglutinin (WGA; W1126: Invitrogen), anti-CD63 monoclonal antibody (MX-49.129.5: sc-5275, Santa Cruz Biotechnology), anti-CD63 monoclonal antibody (cl 3–13: Fuji Film Co.), anti-ARF6 monoclonal antibody (3A-1: sc-7971, Santa Cruz Biotechnology), anti-ARF6 polyclonal rabbit antibody (20225-1-AP, Proteintech), Alexa Fluor 488-conjugated goat-anti-mouse IgG(H+L), F(ab’)2 fragment (#4408, Cell Signaling), Alexa Fluor 488-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4412, Cell Signaling Technology), Alexa Fluor 568-labeled donkey-anti-mouse IgG (H+L) (A10037, Invitrogen), Alexa Fluor 594-labeled goat anti-mouse IgG IgG(H+L), F(ab’)2 fragment (A-11020, Molecular Probes, Inc.), and Alexa Fluor 647-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4414, Cell Signaling Technology).

Techniques: Immunofluorescence, Labeling

Journal: PLoS ONE

Article Title: Subcellular localization of glypican-5 is associated with dynamic motility of the human mesenchymal stem cell line U3DT

doi: 10.1371/journal.pone.0226538

Figure Lengend Snippet: U3DT cells at different stages of mitosis and cytokinesis were fixed but not permeabilized, stained with anti-GPC5 (red) and anti-FGFR1 (green) antibodies, and counterstained with DAPI stain (blue). Yellow represents the degree of colocalization. Scale bar, 5 μm.

Article Snippet: The following antibodies and fluorescence reagents were used: anti-GPC5 antibody (MAB2607, R&D Systems, Inc.), Alexa Fluor 594 conjugated anti-GPC5 antibody (R&D Systems, Inc.), Alexa Fluor 647 conjugated anti-GPC5 antibody (R&D Systems, Inc.), anti-FGFR1 Xp rabbit monoclonal antibody (D8E4, Cell Signaling Technology), anti-Rab11A antibody (A-6: sc-166912, Santa Cruz Biotech), anti-Rab11 (D4F5)XP rabbit monoclonal antibody (Cell Signaling Technology), anti-acetyl-alpha-tubulin rabbit monoclonal antibody (D20G3, #5335, Cell Signaling Technology), Alexa Fluor 488-conjucated wheat germ agglutinin (WGA; W1126: Invitrogen), anti-CD63 monoclonal antibody (MX-49.129.5: sc-5275, Santa Cruz Biotechnology), anti-CD63 monoclonal antibody (cl 3–13: Fuji Film Co.), anti-ARF6 monoclonal antibody (3A-1: sc-7971, Santa Cruz Biotechnology), anti-ARF6 polyclonal rabbit antibody (20225-1-AP, Proteintech), Alexa Fluor 488-conjugated goat-anti-mouse IgG(H+L), F(ab’)2 fragment (#4408, Cell Signaling), Alexa Fluor 488-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4412, Cell Signaling Technology), Alexa Fluor 568-labeled donkey-anti-mouse IgG (H+L) (A10037, Invitrogen), Alexa Fluor 594-labeled goat anti-mouse IgG IgG(H+L), F(ab’)2 fragment (A-11020, Molecular Probes, Inc.), and Alexa Fluor 647-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4414, Cell Signaling Technology).

Techniques: Staining

Journal: PLoS ONE

Article Title: Subcellular localization of glypican-5 is associated with dynamic motility of the human mesenchymal stem cell line U3DT

doi: 10.1371/journal.pone.0226538

Figure Lengend Snippet: (A) Rab11 localized on both sides (red) of the midbody dark zone and partially overlapped with microtubules (gray). Localizations of proteins on midbody microtubules were determined and compared by line scans. Microtubules and Rab11 peaked at the same positions, where the microtubule signal was high and the FGFR signal (green) was low. (B) Rab11 localization (red) adjacent to the midbody. Plasma membrane was stained with WGA (green). (C) GPC5 (red) colocalized with Rab11 (gray) at the midbody. The plasma membrane was stained with WGA (green). (D) GPC5 (red) colocalized with Rab11 (green) on midbody microtubules (gray). Scale bar, 2 μm.

Article Snippet: The following antibodies and fluorescence reagents were used: anti-GPC5 antibody (MAB2607, R&D Systems, Inc.), Alexa Fluor 594 conjugated anti-GPC5 antibody (R&D Systems, Inc.), Alexa Fluor 647 conjugated anti-GPC5 antibody (R&D Systems, Inc.), anti-FGFR1 Xp rabbit monoclonal antibody (D8E4, Cell Signaling Technology), anti-Rab11A antibody (A-6: sc-166912, Santa Cruz Biotech), anti-Rab11 (D4F5)XP rabbit monoclonal antibody (Cell Signaling Technology), anti-acetyl-alpha-tubulin rabbit monoclonal antibody (D20G3, #5335, Cell Signaling Technology), Alexa Fluor 488-conjucated wheat germ agglutinin (WGA; W1126: Invitrogen), anti-CD63 monoclonal antibody (MX-49.129.5: sc-5275, Santa Cruz Biotechnology), anti-CD63 monoclonal antibody (cl 3–13: Fuji Film Co.), anti-ARF6 monoclonal antibody (3A-1: sc-7971, Santa Cruz Biotechnology), anti-ARF6 polyclonal rabbit antibody (20225-1-AP, Proteintech), Alexa Fluor 488-conjugated goat-anti-mouse IgG(H+L), F(ab’)2 fragment (#4408, Cell Signaling), Alexa Fluor 488-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4412, Cell Signaling Technology), Alexa Fluor 568-labeled donkey-anti-mouse IgG (H+L) (A10037, Invitrogen), Alexa Fluor 594-labeled goat anti-mouse IgG IgG(H+L), F(ab’)2 fragment (A-11020, Molecular Probes, Inc.), and Alexa Fluor 647-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4414, Cell Signaling Technology).

Techniques: Clinical Proteomics, Membrane, Staining

Journal: PLoS ONE

Article Title: Subcellular localization of glypican-5 is associated with dynamic motility of the human mesenchymal stem cell line U3DT

doi: 10.1371/journal.pone.0226538

Figure Lengend Snippet: (A‒D) Images of staining with Alexa Fluor 488-conjugated WGA (green) and DAPI (blue) at interphase (A), metaphase (B), and anaphase and telophase (C, D). (D) Maximum projection of 15 Z-staged-images stained with Alexa Fluor 488-WGA and DAPI. (E) U3DT cell at telophase, stained with anti-GPC5 antibody (red), Alexa Fluor 488-WGA (green), and DAPI (blue). (F) Blebs of U3DT cell at telophase stained with anti-FGFR1 (brown), Alexa Fluor 488-WGA (green), and DAPI (blue). (G) Blebs of U3DT cells at telophase stained with anti-Rab11 rabbit antibodies (magenta), Alexa Fluor 488-WGA (green), and DAPI (blue). Scale bar, 5 μm.

Article Snippet: The following antibodies and fluorescence reagents were used: anti-GPC5 antibody (MAB2607, R&D Systems, Inc.), Alexa Fluor 594 conjugated anti-GPC5 antibody (R&D Systems, Inc.), Alexa Fluor 647 conjugated anti-GPC5 antibody (R&D Systems, Inc.), anti-FGFR1 Xp rabbit monoclonal antibody (D8E4, Cell Signaling Technology), anti-Rab11A antibody (A-6: sc-166912, Santa Cruz Biotech), anti-Rab11 (D4F5)XP rabbit monoclonal antibody (Cell Signaling Technology), anti-acetyl-alpha-tubulin rabbit monoclonal antibody (D20G3, #5335, Cell Signaling Technology), Alexa Fluor 488-conjucated wheat germ agglutinin (WGA; W1126: Invitrogen), anti-CD63 monoclonal antibody (MX-49.129.5: sc-5275, Santa Cruz Biotechnology), anti-CD63 monoclonal antibody (cl 3–13: Fuji Film Co.), anti-ARF6 monoclonal antibody (3A-1: sc-7971, Santa Cruz Biotechnology), anti-ARF6 polyclonal rabbit antibody (20225-1-AP, Proteintech), Alexa Fluor 488-conjugated goat-anti-mouse IgG(H+L), F(ab’)2 fragment (#4408, Cell Signaling), Alexa Fluor 488-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4412, Cell Signaling Technology), Alexa Fluor 568-labeled donkey-anti-mouse IgG (H+L) (A10037, Invitrogen), Alexa Fluor 594-labeled goat anti-mouse IgG IgG(H+L), F(ab’)2 fragment (A-11020, Molecular Probes, Inc.), and Alexa Fluor 647-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4414, Cell Signaling Technology).

Techniques: Staining

Journal: PLoS ONE

Article Title: Subcellular localization of glypican-5 is associated with dynamic motility of the human mesenchymal stem cell line U3DT

doi: 10.1371/journal.pone.0226538

Figure Lengend Snippet: Immunofluorescence images of cells were obtained using a Leica SP-8 confocal microscope, and the pixel sum was estimated with the SP8 software (RAS X). (A,B) Images of Rab11 in untreated (A) and RAB11A-siRNA-treated U3DT cells (B). (C) Quantitative analysis of U3DT cells not treated (blue) or treated (red) with 100 nM RAB11A-siRNA. (D,E) Images of GPC5 in U3DT cells not treated (D) or treated (E) with 100 nM RAB11A-siRNA. (F) Quantitative expression of GPC5 in U3DT cells not treated (blue) or treated (red) with 100 nM RAB11A-siRNA. (G,H) Images of GPC5 (red) and Rab11 (green) in trypsinized-U3DT cells not treated (G) or treated (H) with RAB11A-siRNA. (G‒J) Immunofluorescence images (G,H) and the pixel sum (I,J) of trypsinized cells were obtained using a Leica SP-8 immunofluorescence microscope the same as in C and F. (I) The mean intensity (pixel sum) of Rab11 relative to that of DAPI is shown for each untreated (blue) and RAB11A-siRNA-treated U3DT cell (red). (J) The mean fluorescence intensity (pixel sum) of GPC5 relative to that of DAPI staining per cell is shown for each untreated (blue) or RAB11A-siRNA-treated U3DT cell (red). (K‒M) FACS analysis. (K) Contour display of merged three images. U3DT cells were stained with (blue) or without (red) anti-GPC5 and anti-Rab11 antibodies. RAB11A-siRNA-treated cells were stained with anti-GPC5 and anti-Rab11 antibodies (green). (L) Histogram of Alexa Fluor 488 fluorescence intensity (Rab11) of RAB11A-siRNA-treated cells (green) or not treated cells (blue). (M) Histogram of Alexa Fluor 594 fluorescence intensity (GPC5) of RAB11A-siRNA-treated cells (green) or not treated cells (blue). The same preparation was used for immunofluorescence (G, H) and for FACS analyzes (L, M). Red arrows indicate the peak position of immunostaining control cells (L, M). (N, O) Immunofluorescence images of Rab11 (red) and WGA (green) in not treated cells (N) and RAB11A-siRNA-treated cells (O). (P) The mean distribution of telophase cells with three or more blebs (>ca. 2 μm diameter) in telophase are shown for not treated cells (blue) and RAB11A-siRNA-treated cells (red). The numbers in parentheses in Figure (C, F, I, J and P) are the number of images. White arrows indicate blebs in a telophase cell (N). Scale bar, 5 μm.

Article Snippet: The following antibodies and fluorescence reagents were used: anti-GPC5 antibody (MAB2607, R&D Systems, Inc.), Alexa Fluor 594 conjugated anti-GPC5 antibody (R&D Systems, Inc.), Alexa Fluor 647 conjugated anti-GPC5 antibody (R&D Systems, Inc.), anti-FGFR1 Xp rabbit monoclonal antibody (D8E4, Cell Signaling Technology), anti-Rab11A antibody (A-6: sc-166912, Santa Cruz Biotech), anti-Rab11 (D4F5)XP rabbit monoclonal antibody (Cell Signaling Technology), anti-acetyl-alpha-tubulin rabbit monoclonal antibody (D20G3, #5335, Cell Signaling Technology), Alexa Fluor 488-conjucated wheat germ agglutinin (WGA; W1126: Invitrogen), anti-CD63 monoclonal antibody (MX-49.129.5: sc-5275, Santa Cruz Biotechnology), anti-CD63 monoclonal antibody (cl 3–13: Fuji Film Co.), anti-ARF6 monoclonal antibody (3A-1: sc-7971, Santa Cruz Biotechnology), anti-ARF6 polyclonal rabbit antibody (20225-1-AP, Proteintech), Alexa Fluor 488-conjugated goat-anti-mouse IgG(H+L), F(ab’)2 fragment (#4408, Cell Signaling), Alexa Fluor 488-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4412, Cell Signaling Technology), Alexa Fluor 568-labeled donkey-anti-mouse IgG (H+L) (A10037, Invitrogen), Alexa Fluor 594-labeled goat anti-mouse IgG IgG(H+L), F(ab’)2 fragment (A-11020, Molecular Probes, Inc.), and Alexa Fluor 647-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4414, Cell Signaling Technology).

Techniques: Immunofluorescence, Microscopy, Software, Expressing, Fluorescence, Staining, Immunostaining, Control

Journal: PLoS ONE

Article Title: Subcellular localization of glypican-5 is associated with dynamic motility of the human mesenchymal stem cell line U3DT

doi: 10.1371/journal.pone.0226538

Figure Lengend Snippet: (A) Electron microscopic image of negative-stained EVs. The inset shows a magnification of the boxed EV. (B) Size distribution of vesicles in EV preparations measured by image software (n = 110). (C) (F) (I) (L) GPC5-immunostained EVs (red) stained for Rab11 (green) (C), FGFR1 (green) (F), CD63 (green) (I), and ARF6 (green) (L). In (I) and (L), an Alexa Fluor 647- conjugated anti-GPC5 antibody was used to detect of GPC5-ositive particles. (D) (G) (J) (M) Line scan determination of the red bars in (C), (F), (I), and (L): GPC5 (red), others (green). (E) (H) (K) (N) Distribution of GPC5-positive particles identified by scan determinations in (D), (G), (J), and (M). n = 1,516, (E), 630 (H), 573 (K), and 835 (N). Scale bars: (A), 500 nm; (C), (F), (I), (L), 2 μm.

Article Snippet: The following antibodies and fluorescence reagents were used: anti-GPC5 antibody (MAB2607, R&D Systems, Inc.), Alexa Fluor 594 conjugated anti-GPC5 antibody (R&D Systems, Inc.), Alexa Fluor 647 conjugated anti-GPC5 antibody (R&D Systems, Inc.), anti-FGFR1 Xp rabbit monoclonal antibody (D8E4, Cell Signaling Technology), anti-Rab11A antibody (A-6: sc-166912, Santa Cruz Biotech), anti-Rab11 (D4F5)XP rabbit monoclonal antibody (Cell Signaling Technology), anti-acetyl-alpha-tubulin rabbit monoclonal antibody (D20G3, #5335, Cell Signaling Technology), Alexa Fluor 488-conjucated wheat germ agglutinin (WGA; W1126: Invitrogen), anti-CD63 monoclonal antibody (MX-49.129.5: sc-5275, Santa Cruz Biotechnology), anti-CD63 monoclonal antibody (cl 3–13: Fuji Film Co.), anti-ARF6 monoclonal antibody (3A-1: sc-7971, Santa Cruz Biotechnology), anti-ARF6 polyclonal rabbit antibody (20225-1-AP, Proteintech), Alexa Fluor 488-conjugated goat-anti-mouse IgG(H+L), F(ab’)2 fragment (#4408, Cell Signaling), Alexa Fluor 488-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4412, Cell Signaling Technology), Alexa Fluor 568-labeled donkey-anti-mouse IgG (H+L) (A10037, Invitrogen), Alexa Fluor 594-labeled goat anti-mouse IgG IgG(H+L), F(ab’)2 fragment (A-11020, Molecular Probes, Inc.), and Alexa Fluor 647-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4414, Cell Signaling Technology).

Techniques: Staining, Software

Journal: PLoS ONE

Article Title: Subcellular localization of glypican-5 is associated with dynamic motility of the human mesenchymal stem cell line U3DT

doi: 10.1371/journal.pone.0226538

Figure Lengend Snippet: (A, D) Images of control cells. (B, E) Images of EV-treated cells. (D, E) Merged images of UE6E7T-3 cells (D) and EV-treated cells (E) were stained for GPC5 (red) and FGFR1 (green). (C) Quantitation of GPC5 (pixel sum per cell) in control UE6E7T-3 cells (blue) or cells cultured with EVs for 1 day (red). n = 67 (blue) and 78 (red). (F, G) FACS pattern of GPC5 in UE6E7T-3 cells (F) and in cells cultured with EVs for 1 day (G). ⇔ indicates GPC5-positive cells. Scale bar, 5 μm.

Article Snippet: The following antibodies and fluorescence reagents were used: anti-GPC5 antibody (MAB2607, R&D Systems, Inc.), Alexa Fluor 594 conjugated anti-GPC5 antibody (R&D Systems, Inc.), Alexa Fluor 647 conjugated anti-GPC5 antibody (R&D Systems, Inc.), anti-FGFR1 Xp rabbit monoclonal antibody (D8E4, Cell Signaling Technology), anti-Rab11A antibody (A-6: sc-166912, Santa Cruz Biotech), anti-Rab11 (D4F5)XP rabbit monoclonal antibody (Cell Signaling Technology), anti-acetyl-alpha-tubulin rabbit monoclonal antibody (D20G3, #5335, Cell Signaling Technology), Alexa Fluor 488-conjucated wheat germ agglutinin (WGA; W1126: Invitrogen), anti-CD63 monoclonal antibody (MX-49.129.5: sc-5275, Santa Cruz Biotechnology), anti-CD63 monoclonal antibody (cl 3–13: Fuji Film Co.), anti-ARF6 monoclonal antibody (3A-1: sc-7971, Santa Cruz Biotechnology), anti-ARF6 polyclonal rabbit antibody (20225-1-AP, Proteintech), Alexa Fluor 488-conjugated goat-anti-mouse IgG(H+L), F(ab’)2 fragment (#4408, Cell Signaling), Alexa Fluor 488-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4412, Cell Signaling Technology), Alexa Fluor 568-labeled donkey-anti-mouse IgG (H+L) (A10037, Invitrogen), Alexa Fluor 594-labeled goat anti-mouse IgG IgG(H+L), F(ab’)2 fragment (A-11020, Molecular Probes, Inc.), and Alexa Fluor 647-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4414, Cell Signaling Technology).

Techniques: Control, Staining, Quantitation Assay, Cell Culture

Journal: PLoS ONE

Article Title: Subcellular localization of glypican-5 is associated with dynamic motility of the human mesenchymal stem cell line U3DT

doi: 10.1371/journal.pone.0226538

Figure Lengend Snippet: (A) Control cells fixed immediately after scratching. The wide of the scratched area was ca. 500μm. (B‒D) Mock (B), non-targeting siRNA (NT)-treated (C), and GPC5-siRNA (KD)-treated (D) cells were cultured in appropriate medium containing 25 nM FGF2 for 23 h after scratching. Cells were fixed and immunofluorescence images were acquired using a Leica SP-8 microscope equipped with a 20x objective. (E) The number of cells that moved into the scratched area or the removed insert area was counted after incubation of control (without siRNA) cells (yellow), NT cells (red), and KD cells (blue) in appropriate medium containing (red and blue columns) or lacking (brown and sky blue columns) FGF2 at 37°C for 23 h. (F‒H) Immunofluorescence images of cells treated with mock (F), non-targeting siRNA (G), or GPC5-targeting siRNA (H) for 72 h after removing an insert from a μ -Dish. Cells were stained with anti-GPC5 (red) and anti-FGFR1 (green) antibodies. (I) Quantification of the percentage of cells with blebs at telophase. Control (mock) cells (yellow), NT cells (red) and KD cells (blue) were incubated in appropriate medium containing FGF2 at 37°C for 23 h. (J‒L) Immunofluorescence images of mock (J), non-targeting siRNA-treated (K), and GPC5-siRNA-treated (L) cells after 72 h. Cells were stained with WGA (green). White arrows indicate blebs in a telophase cell (J, K). The numbers in parentheses in (E, I) are the number of images observed. Scale bar: (F‒H), 100 μm; (J‒L), 2 μm.

Article Snippet: The following antibodies and fluorescence reagents were used: anti-GPC5 antibody (MAB2607, R&D Systems, Inc.), Alexa Fluor 594 conjugated anti-GPC5 antibody (R&D Systems, Inc.), Alexa Fluor 647 conjugated anti-GPC5 antibody (R&D Systems, Inc.), anti-FGFR1 Xp rabbit monoclonal antibody (D8E4, Cell Signaling Technology), anti-Rab11A antibody (A-6: sc-166912, Santa Cruz Biotech), anti-Rab11 (D4F5)XP rabbit monoclonal antibody (Cell Signaling Technology), anti-acetyl-alpha-tubulin rabbit monoclonal antibody (D20G3, #5335, Cell Signaling Technology), Alexa Fluor 488-conjucated wheat germ agglutinin (WGA; W1126: Invitrogen), anti-CD63 monoclonal antibody (MX-49.129.5: sc-5275, Santa Cruz Biotechnology), anti-CD63 monoclonal antibody (cl 3–13: Fuji Film Co.), anti-ARF6 monoclonal antibody (3A-1: sc-7971, Santa Cruz Biotechnology), anti-ARF6 polyclonal rabbit antibody (20225-1-AP, Proteintech), Alexa Fluor 488-conjugated goat-anti-mouse IgG(H+L), F(ab’)2 fragment (#4408, Cell Signaling), Alexa Fluor 488-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4412, Cell Signaling Technology), Alexa Fluor 568-labeled donkey-anti-mouse IgG (H+L) (A10037, Invitrogen), Alexa Fluor 594-labeled goat anti-mouse IgG IgG(H+L), F(ab’)2 fragment (A-11020, Molecular Probes, Inc.), and Alexa Fluor 647-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4414, Cell Signaling Technology).

Techniques: Control, Cell Culture, Immunofluorescence, Microscopy, Incubation, Staining

Journal: PLoS ONE

Article Title: Subcellular localization of glypican-5 is associated with dynamic motility of the human mesenchymal stem cell line U3DT

doi: 10.1371/journal.pone.0226538

Figure Lengend Snippet: (a) GPC5 (red) localizes at the leading edge of migrating cells, (b) at the equatorial plane, (c) at the furrow, (d) at the intercellular bridge during mitosis, (e) in membrane blebs during cytokinesis, (f) at the tips of filopodia, and (g) in EVs.

Article Snippet: The following antibodies and fluorescence reagents were used: anti-GPC5 antibody (MAB2607, R&D Systems, Inc.), Alexa Fluor 594 conjugated anti-GPC5 antibody (R&D Systems, Inc.), Alexa Fluor 647 conjugated anti-GPC5 antibody (R&D Systems, Inc.), anti-FGFR1 Xp rabbit monoclonal antibody (D8E4, Cell Signaling Technology), anti-Rab11A antibody (A-6: sc-166912, Santa Cruz Biotech), anti-Rab11 (D4F5)XP rabbit monoclonal antibody (Cell Signaling Technology), anti-acetyl-alpha-tubulin rabbit monoclonal antibody (D20G3, #5335, Cell Signaling Technology), Alexa Fluor 488-conjucated wheat germ agglutinin (WGA; W1126: Invitrogen), anti-CD63 monoclonal antibody (MX-49.129.5: sc-5275, Santa Cruz Biotechnology), anti-CD63 monoclonal antibody (cl 3–13: Fuji Film Co.), anti-ARF6 monoclonal antibody (3A-1: sc-7971, Santa Cruz Biotechnology), anti-ARF6 polyclonal rabbit antibody (20225-1-AP, Proteintech), Alexa Fluor 488-conjugated goat-anti-mouse IgG(H+L), F(ab’)2 fragment (#4408, Cell Signaling), Alexa Fluor 488-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4412, Cell Signaling Technology), Alexa Fluor 568-labeled donkey-anti-mouse IgG (H+L) (A10037, Invitrogen), Alexa Fluor 594-labeled goat anti-mouse IgG IgG(H+L), F(ab’)2 fragment (A-11020, Molecular Probes, Inc.), and Alexa Fluor 647-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4414, Cell Signaling Technology).

Techniques: Membrane

Journal: PLoS ONE

Article Title: Subcellular localization of glypican-5 is associated with dynamic motility of the human mesenchymal stem cell line U3DT

doi: 10.1371/journal.pone.0226538

Figure Lengend Snippet: Gene expression of GPCs (GPC1 to GPC6) at four culture stages were analyzed using data in the DDBJ database of the National Institute of Genetics ( http://www.ddbj.nig.ac.jp/ ), accession number: DRA000533 . Each value is shown relative to the corresponding value in U3-A cells (culture stage 1). GPC1, red; GPC2, green; GPC3, no expression; GPC4, purple; GPC5, blue; GPC6, light blue. As shown in the previous report , PDLs of U3-A, U3-B, U3-C and U3DT cells are 60‒90, 91‒150, 151‒230 and 231‒295, respectively.

Article Snippet: The following antibodies and fluorescence reagents were used: anti-GPC5 antibody (MAB2607, R&D Systems, Inc.), Alexa Fluor 594 conjugated anti-GPC5 antibody (R&D Systems, Inc.), Alexa Fluor 647 conjugated anti-GPC5 antibody (R&D Systems, Inc.), anti-FGFR1 Xp rabbit monoclonal antibody (D8E4, Cell Signaling Technology), anti-Rab11A antibody (A-6: sc-166912, Santa Cruz Biotech), anti-Rab11 (D4F5)XP rabbit monoclonal antibody (Cell Signaling Technology), anti-acetyl-alpha-tubulin rabbit monoclonal antibody (D20G3, #5335, Cell Signaling Technology), Alexa Fluor 488-conjucated wheat germ agglutinin (WGA; W1126: Invitrogen), anti-CD63 monoclonal antibody (MX-49.129.5: sc-5275, Santa Cruz Biotechnology), anti-CD63 monoclonal antibody (cl 3–13: Fuji Film Co.), anti-ARF6 monoclonal antibody (3A-1: sc-7971, Santa Cruz Biotechnology), anti-ARF6 polyclonal rabbit antibody (20225-1-AP, Proteintech), Alexa Fluor 488-conjugated goat-anti-mouse IgG(H+L), F(ab’)2 fragment (#4408, Cell Signaling), Alexa Fluor 488-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4412, Cell Signaling Technology), Alexa Fluor 568-labeled donkey-anti-mouse IgG (H+L) (A10037, Invitrogen), Alexa Fluor 594-labeled goat anti-mouse IgG IgG(H+L), F(ab’)2 fragment (A-11020, Molecular Probes, Inc.), and Alexa Fluor 647-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4414, Cell Signaling Technology).

Techniques: Gene Expression, Expressing

Journal: PLoS ONE

Article Title: Subcellular localization of glypican-5 is associated with dynamic motility of the human mesenchymal stem cell line U3DT

doi: 10.1371/journal.pone.0226538

Figure Lengend Snippet: Double immunofluorescence labeling of U3DT cells with anti-GPC5 (red) and anti-FGFR (green) (top three lines), with anti-GPC5 (red) and anti-ARF (green) (fourth line), or with anti-GPC5 (red) and anti-Ra11A (green) (bottom line) antibodies. Positive spots (yellow) in each merged image were clearly visible in U3DT cells. Scale bar, 5 μm.

Article Snippet: The following antibodies and fluorescence reagents were used: anti-GPC5 antibody (MAB2607, R&D Systems, Inc.), Alexa Fluor 594 conjugated anti-GPC5 antibody (R&D Systems, Inc.), Alexa Fluor 647 conjugated anti-GPC5 antibody (R&D Systems, Inc.), anti-FGFR1 Xp rabbit monoclonal antibody (D8E4, Cell Signaling Technology), anti-Rab11A antibody (A-6: sc-166912, Santa Cruz Biotech), anti-Rab11 (D4F5)XP rabbit monoclonal antibody (Cell Signaling Technology), anti-acetyl-alpha-tubulin rabbit monoclonal antibody (D20G3, #5335, Cell Signaling Technology), Alexa Fluor 488-conjucated wheat germ agglutinin (WGA; W1126: Invitrogen), anti-CD63 monoclonal antibody (MX-49.129.5: sc-5275, Santa Cruz Biotechnology), anti-CD63 monoclonal antibody (cl 3–13: Fuji Film Co.), anti-ARF6 monoclonal antibody (3A-1: sc-7971, Santa Cruz Biotechnology), anti-ARF6 polyclonal rabbit antibody (20225-1-AP, Proteintech), Alexa Fluor 488-conjugated goat-anti-mouse IgG(H+L), F(ab’)2 fragment (#4408, Cell Signaling), Alexa Fluor 488-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4412, Cell Signaling Technology), Alexa Fluor 568-labeled donkey-anti-mouse IgG (H+L) (A10037, Invitrogen), Alexa Fluor 594-labeled goat anti-mouse IgG IgG(H+L), F(ab’)2 fragment (A-11020, Molecular Probes, Inc.), and Alexa Fluor 647-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4414, Cell Signaling Technology).

Techniques: Immunofluorescence, Labeling

Journal: PLoS ONE

Article Title: Subcellular localization of glypican-5 is associated with dynamic motility of the human mesenchymal stem cell line U3DT

doi: 10.1371/journal.pone.0226538

Figure Lengend Snippet: U3DT cells at different stages of mitosis and cytokinesis were fixed but not permeabilized, stained with anti-GPC5 (red) and anti-FGFR1 (green) antibodies, and counterstained with DAPI stain (blue). Yellow represents the degree of colocalization. Scale bar, 5 μm.

Article Snippet: The following antibodies and fluorescence reagents were used: anti-GPC5 antibody (MAB2607, R&D Systems, Inc.), Alexa Fluor 594 conjugated anti-GPC5 antibody (R&D Systems, Inc.), Alexa Fluor 647 conjugated anti-GPC5 antibody (R&D Systems, Inc.), anti-FGFR1 Xp rabbit monoclonal antibody (D8E4, Cell Signaling Technology), anti-Rab11A antibody (A-6: sc-166912, Santa Cruz Biotech), anti-Rab11 (D4F5)XP rabbit monoclonal antibody (Cell Signaling Technology), anti-acetyl-alpha-tubulin rabbit monoclonal antibody (D20G3, #5335, Cell Signaling Technology), Alexa Fluor 488-conjucated wheat germ agglutinin (WGA; W1126: Invitrogen), anti-CD63 monoclonal antibody (MX-49.129.5: sc-5275, Santa Cruz Biotechnology), anti-CD63 monoclonal antibody (cl 3–13: Fuji Film Co.), anti-ARF6 monoclonal antibody (3A-1: sc-7971, Santa Cruz Biotechnology), anti-ARF6 polyclonal rabbit antibody (20225-1-AP, Proteintech), Alexa Fluor 488-conjugated goat-anti-mouse IgG(H+L), F(ab’)2 fragment (#4408, Cell Signaling), Alexa Fluor 488-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4412, Cell Signaling Technology), Alexa Fluor 568-labeled donkey-anti-mouse IgG (H+L) (A10037, Invitrogen), Alexa Fluor 594-labeled goat anti-mouse IgG IgG(H+L), F(ab’)2 fragment (A-11020, Molecular Probes, Inc.), and Alexa Fluor 647-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4414, Cell Signaling Technology).

Techniques: Staining

Journal: PLoS ONE

Article Title: Subcellular localization of glypican-5 is associated with dynamic motility of the human mesenchymal stem cell line U3DT

doi: 10.1371/journal.pone.0226538

Figure Lengend Snippet: (A) Rab11 localized on both sides (red) of the midbody dark zone and partially overlapped with microtubules (gray). Localizations of proteins on midbody microtubules were determined and compared by line scans. Microtubules and Rab11 peaked at the same positions, where the microtubule signal was high and the FGFR signal (green) was low. (B) Rab11 localization (red) adjacent to the midbody. Plasma membrane was stained with WGA (green). (C) GPC5 (red) colocalized with Rab11 (gray) at the midbody. The plasma membrane was stained with WGA (green). (D) GPC5 (red) colocalized with Rab11 (green) on midbody microtubules (gray). Scale bar, 2 μm.

Article Snippet: The following antibodies and fluorescence reagents were used: anti-GPC5 antibody (MAB2607, R&D Systems, Inc.), Alexa Fluor 594 conjugated anti-GPC5 antibody (R&D Systems, Inc.), Alexa Fluor 647 conjugated anti-GPC5 antibody (R&D Systems, Inc.), anti-FGFR1 Xp rabbit monoclonal antibody (D8E4, Cell Signaling Technology), anti-Rab11A antibody (A-6: sc-166912, Santa Cruz Biotech), anti-Rab11 (D4F5)XP rabbit monoclonal antibody (Cell Signaling Technology), anti-acetyl-alpha-tubulin rabbit monoclonal antibody (D20G3, #5335, Cell Signaling Technology), Alexa Fluor 488-conjucated wheat germ agglutinin (WGA; W1126: Invitrogen), anti-CD63 monoclonal antibody (MX-49.129.5: sc-5275, Santa Cruz Biotechnology), anti-CD63 monoclonal antibody (cl 3–13: Fuji Film Co.), anti-ARF6 monoclonal antibody (3A-1: sc-7971, Santa Cruz Biotechnology), anti-ARF6 polyclonal rabbit antibody (20225-1-AP, Proteintech), Alexa Fluor 488-conjugated goat-anti-mouse IgG(H+L), F(ab’)2 fragment (#4408, Cell Signaling), Alexa Fluor 488-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4412, Cell Signaling Technology), Alexa Fluor 568-labeled donkey-anti-mouse IgG (H+L) (A10037, Invitrogen), Alexa Fluor 594-labeled goat anti-mouse IgG IgG(H+L), F(ab’)2 fragment (A-11020, Molecular Probes, Inc.), and Alexa Fluor 647-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4414, Cell Signaling Technology).

Techniques: Clinical Proteomics, Membrane, Staining

Journal: PLoS ONE

Article Title: Subcellular localization of glypican-5 is associated with dynamic motility of the human mesenchymal stem cell line U3DT

doi: 10.1371/journal.pone.0226538

Figure Lengend Snippet: (A‒D) Images of staining with Alexa Fluor 488-conjugated WGA (green) and DAPI (blue) at interphase (A), metaphase (B), and anaphase and telophase (C, D). (D) Maximum projection of 15 Z-staged-images stained with Alexa Fluor 488-WGA and DAPI. (E) U3DT cell at telophase, stained with anti-GPC5 antibody (red), Alexa Fluor 488-WGA (green), and DAPI (blue). (F) Blebs of U3DT cell at telophase stained with anti-FGFR1 (brown), Alexa Fluor 488-WGA (green), and DAPI (blue). (G) Blebs of U3DT cells at telophase stained with anti-Rab11 rabbit antibodies (magenta), Alexa Fluor 488-WGA (green), and DAPI (blue). Scale bar, 5 μm.

Article Snippet: The following antibodies and fluorescence reagents were used: anti-GPC5 antibody (MAB2607, R&D Systems, Inc.), Alexa Fluor 594 conjugated anti-GPC5 antibody (R&D Systems, Inc.), Alexa Fluor 647 conjugated anti-GPC5 antibody (R&D Systems, Inc.), anti-FGFR1 Xp rabbit monoclonal antibody (D8E4, Cell Signaling Technology), anti-Rab11A antibody (A-6: sc-166912, Santa Cruz Biotech), anti-Rab11 (D4F5)XP rabbit monoclonal antibody (Cell Signaling Technology), anti-acetyl-alpha-tubulin rabbit monoclonal antibody (D20G3, #5335, Cell Signaling Technology), Alexa Fluor 488-conjucated wheat germ agglutinin (WGA; W1126: Invitrogen), anti-CD63 monoclonal antibody (MX-49.129.5: sc-5275, Santa Cruz Biotechnology), anti-CD63 monoclonal antibody (cl 3–13: Fuji Film Co.), anti-ARF6 monoclonal antibody (3A-1: sc-7971, Santa Cruz Biotechnology), anti-ARF6 polyclonal rabbit antibody (20225-1-AP, Proteintech), Alexa Fluor 488-conjugated goat-anti-mouse IgG(H+L), F(ab’)2 fragment (#4408, Cell Signaling), Alexa Fluor 488-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4412, Cell Signaling Technology), Alexa Fluor 568-labeled donkey-anti-mouse IgG (H+L) (A10037, Invitrogen), Alexa Fluor 594-labeled goat anti-mouse IgG IgG(H+L), F(ab’)2 fragment (A-11020, Molecular Probes, Inc.), and Alexa Fluor 647-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4414, Cell Signaling Technology).

Techniques: Staining

Journal: PLoS ONE

Article Title: Subcellular localization of glypican-5 is associated with dynamic motility of the human mesenchymal stem cell line U3DT

doi: 10.1371/journal.pone.0226538

Figure Lengend Snippet: Immunofluorescence images of cells were obtained using a Leica SP-8 confocal microscope, and the pixel sum was estimated with the SP8 software (RAS X). (A,B) Images of Rab11 in untreated (A) and RAB11A-siRNA-treated U3DT cells (B). (C) Quantitative analysis of U3DT cells not treated (blue) or treated (red) with 100 nM RAB11A-siRNA. (D,E) Images of GPC5 in U3DT cells not treated (D) or treated (E) with 100 nM RAB11A-siRNA. (F) Quantitative expression of GPC5 in U3DT cells not treated (blue) or treated (red) with 100 nM RAB11A-siRNA. (G,H) Images of GPC5 (red) and Rab11 (green) in trypsinized-U3DT cells not treated (G) or treated (H) with RAB11A-siRNA. (G‒J) Immunofluorescence images (G,H) and the pixel sum (I,J) of trypsinized cells were obtained using a Leica SP-8 immunofluorescence microscope the same as in C and F. (I) The mean intensity (pixel sum) of Rab11 relative to that of DAPI is shown for each untreated (blue) and RAB11A-siRNA-treated U3DT cell (red). (J) The mean fluorescence intensity (pixel sum) of GPC5 relative to that of DAPI staining per cell is shown for each untreated (blue) or RAB11A-siRNA-treated U3DT cell (red). (K‒M) FACS analysis. (K) Contour display of merged three images. U3DT cells were stained with (blue) or without (red) anti-GPC5 and anti-Rab11 antibodies. RAB11A-siRNA-treated cells were stained with anti-GPC5 and anti-Rab11 antibodies (green). (L) Histogram of Alexa Fluor 488 fluorescence intensity (Rab11) of RAB11A-siRNA-treated cells (green) or not treated cells (blue). (M) Histogram of Alexa Fluor 594 fluorescence intensity (GPC5) of RAB11A-siRNA-treated cells (green) or not treated cells (blue). The same preparation was used for immunofluorescence (G, H) and for FACS analyzes (L, M). Red arrows indicate the peak position of immunostaining control cells (L, M). (N, O) Immunofluorescence images of Rab11 (red) and WGA (green) in not treated cells (N) and RAB11A-siRNA-treated cells (O). (P) The mean distribution of telophase cells with three or more blebs (>ca. 2 μm diameter) in telophase are shown for not treated cells (blue) and RAB11A-siRNA-treated cells (red). The numbers in parentheses in Figure (C, F, I, J and P) are the number of images. White arrows indicate blebs in a telophase cell (N). Scale bar, 5 μm.

Article Snippet: The following antibodies and fluorescence reagents were used: anti-GPC5 antibody (MAB2607, R&D Systems, Inc.), Alexa Fluor 594 conjugated anti-GPC5 antibody (R&D Systems, Inc.), Alexa Fluor 647 conjugated anti-GPC5 antibody (R&D Systems, Inc.), anti-FGFR1 Xp rabbit monoclonal antibody (D8E4, Cell Signaling Technology), anti-Rab11A antibody (A-6: sc-166912, Santa Cruz Biotech), anti-Rab11 (D4F5)XP rabbit monoclonal antibody (Cell Signaling Technology), anti-acetyl-alpha-tubulin rabbit monoclonal antibody (D20G3, #5335, Cell Signaling Technology), Alexa Fluor 488-conjucated wheat germ agglutinin (WGA; W1126: Invitrogen), anti-CD63 monoclonal antibody (MX-49.129.5: sc-5275, Santa Cruz Biotechnology), anti-CD63 monoclonal antibody (cl 3–13: Fuji Film Co.), anti-ARF6 monoclonal antibody (3A-1: sc-7971, Santa Cruz Biotechnology), anti-ARF6 polyclonal rabbit antibody (20225-1-AP, Proteintech), Alexa Fluor 488-conjugated goat-anti-mouse IgG(H+L), F(ab’)2 fragment (#4408, Cell Signaling), Alexa Fluor 488-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4412, Cell Signaling Technology), Alexa Fluor 568-labeled donkey-anti-mouse IgG (H+L) (A10037, Invitrogen), Alexa Fluor 594-labeled goat anti-mouse IgG IgG(H+L), F(ab’)2 fragment (A-11020, Molecular Probes, Inc.), and Alexa Fluor 647-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4414, Cell Signaling Technology).

Techniques: Immunofluorescence, Microscopy, Software, Expressing, Fluorescence, Staining, Immunostaining, Control

Journal: PLoS ONE

Article Title: Subcellular localization of glypican-5 is associated with dynamic motility of the human mesenchymal stem cell line U3DT

doi: 10.1371/journal.pone.0226538

Figure Lengend Snippet: (A) Electron microscopic image of negative-stained EVs. The inset shows a magnification of the boxed EV. (B) Size distribution of vesicles in EV preparations measured by image software (n = 110). (C) (F) (I) (L) GPC5-immunostained EVs (red) stained for Rab11 (green) (C), FGFR1 (green) (F), CD63 (green) (I), and ARF6 (green) (L). In (I) and (L), an Alexa Fluor 647- conjugated anti-GPC5 antibody was used to detect of GPC5-ositive particles. (D) (G) (J) (M) Line scan determination of the red bars in (C), (F), (I), and (L): GPC5 (red), others (green). (E) (H) (K) (N) Distribution of GPC5-positive particles identified by scan determinations in (D), (G), (J), and (M). n = 1,516, (E), 630 (H), 573 (K), and 835 (N). Scale bars: (A), 500 nm; (C), (F), (I), (L), 2 μm.

Article Snippet: The following antibodies and fluorescence reagents were used: anti-GPC5 antibody (MAB2607, R&D Systems, Inc.), Alexa Fluor 594 conjugated anti-GPC5 antibody (R&D Systems, Inc.), Alexa Fluor 647 conjugated anti-GPC5 antibody (R&D Systems, Inc.), anti-FGFR1 Xp rabbit monoclonal antibody (D8E4, Cell Signaling Technology), anti-Rab11A antibody (A-6: sc-166912, Santa Cruz Biotech), anti-Rab11 (D4F5)XP rabbit monoclonal antibody (Cell Signaling Technology), anti-acetyl-alpha-tubulin rabbit monoclonal antibody (D20G3, #5335, Cell Signaling Technology), Alexa Fluor 488-conjucated wheat germ agglutinin (WGA; W1126: Invitrogen), anti-CD63 monoclonal antibody (MX-49.129.5: sc-5275, Santa Cruz Biotechnology), anti-CD63 monoclonal antibody (cl 3–13: Fuji Film Co.), anti-ARF6 monoclonal antibody (3A-1: sc-7971, Santa Cruz Biotechnology), anti-ARF6 polyclonal rabbit antibody (20225-1-AP, Proteintech), Alexa Fluor 488-conjugated goat-anti-mouse IgG(H+L), F(ab’)2 fragment (#4408, Cell Signaling), Alexa Fluor 488-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4412, Cell Signaling Technology), Alexa Fluor 568-labeled donkey-anti-mouse IgG (H+L) (A10037, Invitrogen), Alexa Fluor 594-labeled goat anti-mouse IgG IgG(H+L), F(ab’)2 fragment (A-11020, Molecular Probes, Inc.), and Alexa Fluor 647-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4414, Cell Signaling Technology).

Techniques: Staining, Software

Journal: PLoS ONE

Article Title: Subcellular localization of glypican-5 is associated with dynamic motility of the human mesenchymal stem cell line U3DT

doi: 10.1371/journal.pone.0226538

Figure Lengend Snippet: (A, D) Images of control cells. (B, E) Images of EV-treated cells. (D, E) Merged images of UE6E7T-3 cells (D) and EV-treated cells (E) were stained for GPC5 (red) and FGFR1 (green). (C) Quantitation of GPC5 (pixel sum per cell) in control UE6E7T-3 cells (blue) or cells cultured with EVs for 1 day (red). n = 67 (blue) and 78 (red). (F, G) FACS pattern of GPC5 in UE6E7T-3 cells (F) and in cells cultured with EVs for 1 day (G). ⇔ indicates GPC5-positive cells. Scale bar, 5 μm.

Article Snippet: The following antibodies and fluorescence reagents were used: anti-GPC5 antibody (MAB2607, R&D Systems, Inc.), Alexa Fluor 594 conjugated anti-GPC5 antibody (R&D Systems, Inc.), Alexa Fluor 647 conjugated anti-GPC5 antibody (R&D Systems, Inc.), anti-FGFR1 Xp rabbit monoclonal antibody (D8E4, Cell Signaling Technology), anti-Rab11A antibody (A-6: sc-166912, Santa Cruz Biotech), anti-Rab11 (D4F5)XP rabbit monoclonal antibody (Cell Signaling Technology), anti-acetyl-alpha-tubulin rabbit monoclonal antibody (D20G3, #5335, Cell Signaling Technology), Alexa Fluor 488-conjucated wheat germ agglutinin (WGA; W1126: Invitrogen), anti-CD63 monoclonal antibody (MX-49.129.5: sc-5275, Santa Cruz Biotechnology), anti-CD63 monoclonal antibody (cl 3–13: Fuji Film Co.), anti-ARF6 monoclonal antibody (3A-1: sc-7971, Santa Cruz Biotechnology), anti-ARF6 polyclonal rabbit antibody (20225-1-AP, Proteintech), Alexa Fluor 488-conjugated goat-anti-mouse IgG(H+L), F(ab’)2 fragment (#4408, Cell Signaling), Alexa Fluor 488-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4412, Cell Signaling Technology), Alexa Fluor 568-labeled donkey-anti-mouse IgG (H+L) (A10037, Invitrogen), Alexa Fluor 594-labeled goat anti-mouse IgG IgG(H+L), F(ab’)2 fragment (A-11020, Molecular Probes, Inc.), and Alexa Fluor 647-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4414, Cell Signaling Technology).

Techniques: Control, Staining, Quantitation Assay, Cell Culture

Journal: PLoS ONE

Article Title: Subcellular localization of glypican-5 is associated with dynamic motility of the human mesenchymal stem cell line U3DT

doi: 10.1371/journal.pone.0226538

Figure Lengend Snippet: (A) Control cells fixed immediately after scratching. The wide of the scratched area was ca. 500μm. (B‒D) Mock (B), non-targeting siRNA (NT)-treated (C), and GPC5-siRNA (KD)-treated (D) cells were cultured in appropriate medium containing 25 nM FGF2 for 23 h after scratching. Cells were fixed and immunofluorescence images were acquired using a Leica SP-8 microscope equipped with a 20x objective. (E) The number of cells that moved into the scratched area or the removed insert area was counted after incubation of control (without siRNA) cells (yellow), NT cells (red), and KD cells (blue) in appropriate medium containing (red and blue columns) or lacking (brown and sky blue columns) FGF2 at 37°C for 23 h. (F‒H) Immunofluorescence images of cells treated with mock (F), non-targeting siRNA (G), or GPC5-targeting siRNA (H) for 72 h after removing an insert from a μ -Dish. Cells were stained with anti-GPC5 (red) and anti-FGFR1 (green) antibodies. (I) Quantification of the percentage of cells with blebs at telophase. Control (mock) cells (yellow), NT cells (red) and KD cells (blue) were incubated in appropriate medium containing FGF2 at 37°C for 23 h. (J‒L) Immunofluorescence images of mock (J), non-targeting siRNA-treated (K), and GPC5-siRNA-treated (L) cells after 72 h. Cells were stained with WGA (green). White arrows indicate blebs in a telophase cell (J, K). The numbers in parentheses in (E, I) are the number of images observed. Scale bar: (F‒H), 100 μm; (J‒L), 2 μm.

Article Snippet: The following antibodies and fluorescence reagents were used: anti-GPC5 antibody (MAB2607, R&D Systems, Inc.), Alexa Fluor 594 conjugated anti-GPC5 antibody (R&D Systems, Inc.), Alexa Fluor 647 conjugated anti-GPC5 antibody (R&D Systems, Inc.), anti-FGFR1 Xp rabbit monoclonal antibody (D8E4, Cell Signaling Technology), anti-Rab11A antibody (A-6: sc-166912, Santa Cruz Biotech), anti-Rab11 (D4F5)XP rabbit monoclonal antibody (Cell Signaling Technology), anti-acetyl-alpha-tubulin rabbit monoclonal antibody (D20G3, #5335, Cell Signaling Technology), Alexa Fluor 488-conjucated wheat germ agglutinin (WGA; W1126: Invitrogen), anti-CD63 monoclonal antibody (MX-49.129.5: sc-5275, Santa Cruz Biotechnology), anti-CD63 monoclonal antibody (cl 3–13: Fuji Film Co.), anti-ARF6 monoclonal antibody (3A-1: sc-7971, Santa Cruz Biotechnology), anti-ARF6 polyclonal rabbit antibody (20225-1-AP, Proteintech), Alexa Fluor 488-conjugated goat-anti-mouse IgG(H+L), F(ab’)2 fragment (#4408, Cell Signaling), Alexa Fluor 488-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4412, Cell Signaling Technology), Alexa Fluor 568-labeled donkey-anti-mouse IgG (H+L) (A10037, Invitrogen), Alexa Fluor 594-labeled goat anti-mouse IgG IgG(H+L), F(ab’)2 fragment (A-11020, Molecular Probes, Inc.), and Alexa Fluor 647-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4414, Cell Signaling Technology).

Techniques: Control, Cell Culture, Immunofluorescence, Microscopy, Incubation, Staining

Journal: PLoS ONE

Article Title: Subcellular localization of glypican-5 is associated with dynamic motility of the human mesenchymal stem cell line U3DT

doi: 10.1371/journal.pone.0226538

Figure Lengend Snippet: (a) GPC5 (red) localizes at the leading edge of migrating cells, (b) at the equatorial plane, (c) at the furrow, (d) at the intercellular bridge during mitosis, (e) in membrane blebs during cytokinesis, (f) at the tips of filopodia, and (g) in EVs.

Article Snippet: The following antibodies and fluorescence reagents were used: anti-GPC5 antibody (MAB2607, R&D Systems, Inc.), Alexa Fluor 594 conjugated anti-GPC5 antibody (R&D Systems, Inc.), Alexa Fluor 647 conjugated anti-GPC5 antibody (R&D Systems, Inc.), anti-FGFR1 Xp rabbit monoclonal antibody (D8E4, Cell Signaling Technology), anti-Rab11A antibody (A-6: sc-166912, Santa Cruz Biotech), anti-Rab11 (D4F5)XP rabbit monoclonal antibody (Cell Signaling Technology), anti-acetyl-alpha-tubulin rabbit monoclonal antibody (D20G3, #5335, Cell Signaling Technology), Alexa Fluor 488-conjucated wheat germ agglutinin (WGA; W1126: Invitrogen), anti-CD63 monoclonal antibody (MX-49.129.5: sc-5275, Santa Cruz Biotechnology), anti-CD63 monoclonal antibody (cl 3–13: Fuji Film Co.), anti-ARF6 monoclonal antibody (3A-1: sc-7971, Santa Cruz Biotechnology), anti-ARF6 polyclonal rabbit antibody (20225-1-AP, Proteintech), Alexa Fluor 488-conjugated goat-anti-mouse IgG(H+L), F(ab’)2 fragment (#4408, Cell Signaling), Alexa Fluor 488-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4412, Cell Signaling Technology), Alexa Fluor 568-labeled donkey-anti-mouse IgG (H+L) (A10037, Invitrogen), Alexa Fluor 594-labeled goat anti-mouse IgG IgG(H+L), F(ab’)2 fragment (A-11020, Molecular Probes, Inc.), and Alexa Fluor 647-conjugated goat-anti-rabbit IgG(H+L), F(ab’)2 fragment (#4414, Cell Signaling Technology).

Techniques: Membrane