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: N-cadherin mechanosensing in ovarian follicles controls oocyte maturation and ovulation

doi: 10.1101/2023.10.06.561232

Figure Lengend Snippet: (a) top panel: Chemical structures of N-cadherin antagonists CRS-066; LCRF-0006 and analogues RB-192 and RB-028. Active side-chain or modified side-chain depicted by highlighted box. middle panel: Average cell adhesion index values of pre-ovulatory COCs (11h post-hCG) interacting with a fibronectin substrate in presence of vehicle or N-Cadherin antagonists CRS-066, LCRF-0006, and sidechain modified analogues RB-192 and RB-028 or vehicle at respective doses. Cell indices were determined using the xCELLigence Impedance system over 15h (n=3 independent experiments with 2 technical replicates per treatment). bottom panel: Mean ± SD adhesion index values at 6 h and IC 50 values of respective drugs calculated from dose-response curve and compared using one-way ANOVA. ( b and d) Representative confocal images of N-cadherin adherens junctions on SK-OV-3 cells treated with N-cadherin antagonists CRS-066 (0.1-1.0 μM), LCRF-0006 (36-360 μM) or vehicle for 24h. (N=3). Scale bar: 40μM.( c and e ) Quantification of N-cadherin adherens junctions. Mean of total pixel intensity in red channel, +/- SEM of at least 50 cells. N=3 independent experiments. Statistical analyses with two-tailed unpaired t-test. * denotes <0.01.( f ) Bright-field images of spheroid formation in 67NR mouse mammary cell line expressing ectopic Cdh2 were treated with CRS-066 or vehicle at respective time-points. Cells were seeded at 2000 cells per well, and formation of spheroids was assessed by imaging every hour for 6h. ( g ) Mean +/- SEM of spheroid area in 67NR-Cdh2 cells treated with either vehicle or increasing doses of CRS-066 (0-2 μM) over 6h.

Article Snippet: Sections were probed with primary antibodies against N-cadherin (BD Biosciences; Cat# 610920; 1:500); β-catenin (CST; Cat#8480; 1:500), E-cadherin (CST, Cat#14472; 1:500); a-tubulin (ThermoFisher; Cat#236-10501); Ki-67 (CST; Cat#9449; 1:1000) and Cleaved caspase 3 (CST; Cat#9661; 1:1000) diluted in 10% NGS and incubated O/N at at 4°C overnight in a humid chamber.

Techniques: Analogues, Modification, Two Tailed Test, Expressing, Imaging

Journal: bioRxiv

Article Title: N-cadherin mechanosensing in ovarian follicles controls oocyte maturation and ovulation

doi: 10.1101/2023.10.06.561232

Figure Lengend Snippet: ( a - c ) Time-course of Cdh2, ctnnb1 and Cdh1 mRNA expression in isolated granulosa cell (GCs) or Cumulus oocyte complexes (COCs) from mouse ovaries at indicated time-points after eCG and hCG stimulation of folliculogenesis and ovulation (N=3 animals per time point). Cdh2 and Ctnnb1 levels are high in GC and COC throughout folliculogenesis, with a transient drop in Cdh2 level 12 h after ovulation stimulus, while E-Cadherin was high in COCs and significantly reduced by ovulation stimulus. The levels shown of the indicated mRNAs were determined by TaqMan qPCR normalised to Rpl19. ( d ) Immunofluorescent staining of N-Cadherin, βcatenin and E-cadherin throughout ovarian folliculogenesis. Confocal images of mouse ovarian sections obtained from eCG primed mice and stained using anti N-Cadherin (left panel), anti-β-catenin (middle panel) and E-cadherin (right panel). DNA is counterstained with Hoechst. Arrows indicate presence of N-Cadherin and β-catenin at granulosa-granulosa cell junctions in secondary and antral follicle stages. Arrowheads indicate presence of N-cadherin and β-catenin at oocyte-cumulus interface. High magnification images show transzonal projections extending from cumulus cells and anchored to oocyte membrane Scale bar: 50 µM. ( e ) Whole-mount immunofluorescent staining showing co-localization of N-cadherin (green) and E-cadherin (red) at the oocyte plasma membrane in mouse COC from antral follicles of eCG primed mice. N-cadherin is also evident on cumulus cell surfaces and transzonal projections. Cumulus cell and oocyte nuclear DNA is counterstained with Hoechst. Scale bar: 50 µM. ( f ) Wholemount immunostaining shows loss of β-catenin and E-cadherin at the oocyte plasma membrane after treatment with CRS-066. COCs obtained from antral follicles of eCG primed mice and treated with CRS-066 or vehicle for 4h. COCs were fixed and stained with anti-E-cadherin (green) and anti-β-catenin (red). DNA was counterstained with Hoechst. Scale bar: 50 µM.

Article Snippet: Sections were probed with primary antibodies against N-cadherin (BD Biosciences; Cat# 610920; 1:500); β-catenin (CST; Cat#8480; 1:500), E-cadherin (CST, Cat#14472; 1:500); a-tubulin (ThermoFisher; Cat#236-10501); Ki-67 (CST; Cat#9449; 1:1000) and Cleaved caspase 3 (CST; Cat#9661; 1:1000) diluted in 10% NGS and incubated O/N at at 4°C overnight in a humid chamber.

Techniques: Expressing, Isolation, Staining, Membrane, Clinical Proteomics, Immunostaining

Journal: bioRxiv

Article Title: N-cadherin mechanosensing in ovarian follicles controls oocyte maturation and ovulation

doi: 10.1101/2023.10.06.561232

Figure Lengend Snippet: COCs from eCG primed mice were treated with LCRF-0006 (36-360 uM) or CRS-066 (0,1-1 uM) during in vitro maturation (EGF and FSH stimulated) and cumulus expansion was assessed after 12 h or gene expression assessed after 10 h IVM. ( a and d ) Representative bright-field images of COCs after 12 h IVM treated with LCRF-0006 or CRS-066. Scale bar: 10µm. ( b and e ) Mean ±SEM of Cumulus expansion indices from a and d n= >20 COCs per experiment. N=4 independent experiments, * = P<0.05, ** = P<0.01. ( c and f ) Effect of N-cadherin antagonist treatment during IVM (10 h) on the expression of key genes involved in COC expansion during IVM. Mean± SEM. N=3 independent experiments. Statistical testing with one-way ANOVA * P<0.05, **P<0.01. ( g and h ) Gene ontology enrichment of biological pathways and molecular functions of significantly differentially down-regulated genes identified in RNA-Seq analysis of COCs after CRS-066 (0.3 μM) treatment compared to vehicle treatment.. All data are presented as the ratio of CRS-066 over vehicle (N=3). ( I and j ) Gene set enrichment analysis plot (GSEA) demonstrating the upregulation of Ctnnb1 and Hippo signalling pathways in CRS-066 treated COCs versus vehicle treated COCs. Net enrichment score (NES) values are shown. N=3 independent biological replicates. ( k ) Heat map representing the relative expression profiles of transcripts involved in Wnt\β- catenin, Hippo\YAP and ovarian signalling axes.

Article Snippet: Sections were probed with primary antibodies against N-cadherin (BD Biosciences; Cat# 610920; 1:500); β-catenin (CST; Cat#8480; 1:500), E-cadherin (CST, Cat#14472; 1:500); a-tubulin (ThermoFisher; Cat#236-10501); Ki-67 (CST; Cat#9449; 1:1000) and Cleaved caspase 3 (CST; Cat#9661; 1:1000) diluted in 10% NGS and incubated O/N at at 4°C overnight in a humid chamber.

Techniques: In Vitro, Gene Expression, Expressing, RNA Sequencing

Journal: bioRxiv

Article Title: N-cadherin mechanosensing in ovarian follicles controls oocyte maturation and ovulation

doi: 10.1101/2023.10.06.561232

Figure Lengend Snippet: ( a ) Ovulation rate of 21d old mice treated with CRS-066 (50mg/kg) or vehicle (7.5% DMSO in 0.9% saline). COCs in oviducts counted 16h after hCG injection. Graph represents mean ±SEM from N=6 animals; ***p<0001 (unpaired two-tailed t-test). ( b ) Histology of ovaries by Haematoxylin & Eosin staining. CL indicate corpus leuteum; arrows indicate trapped oocytes in CL. Scale bar: 100µm. ( c ) Hierarchical clustering of RNA-sequencing analysis results shows differentially expressed genes between CRS-066 and vehicle treated mice (N=6 mice per treatment). ( d and e ) Gene ontology enrichment of biological pathways (D) and molecular functions (E) of significantly differently down-regulated genes in CRS-066 treated mouse ovaries compared to vehicle treated ovaries. ( f and g ) Gene set enrichment analyses plot (GSEA) shows downregulation of Ctnnb1 and Hippo signalling pathways in CRS-066 treated mice ovaries compared to vehicle treated ovaries. Net enrichment score (NES) values are shown. ( h ) Heatmap representing the relative expression profiles of transcripts involved in Wnt\β-catenin, Hippo\YAP and ovarian signalling axes in CRS-066 treated ovaries compared to vehicle. N=3 biological replicates. ( i - k ) Relative mRNA expression of key genes involved in gonadotrophin signalling and oocyte function (i), COC expansion and ovulation (j) or folliculogenesis (k) h in ovaries treated with CRS-066 compared to vehicle treatment and determined by qRT-PCR. Bar graph show mean+/- SEM. N=6 ovaries from independent CRS or vehicle treated mice. Statistical testing with Student’s t-test; *P < 0.05; ** P<0.01; **** P <0.00001. ( l ) Follicle counts at primary, secondary, pre-antral, antral and ovulatory stages in ovaries from mice treated with either CRS-006 (50mg/kg) or vehicle control (7.5% DMSO). N=3 mice/treatment/time-point. ( m ) Representative follicle morphology H&E (left) section and N-cadherin immunofluorescence (right) section in mice treated with either CRS-066 or vehicle. H&E and immunoflourescence highlight disorganised granulosa cells organisation. Asterisks indicate loss of transzonal projections between oocyte and cumulus cells. Scale bar: 30µm. ( n ) Representative confocal immunofluorescent images of mouse ovaries stained with anti-cleaved caspase 3 and anti-Ki-67. Scale bar: 30µm. ( o ) Relative mRNA expression of key genes involved in oocyte growth and ovulation in CRS-066 or vehicle treated mice (N=3/ treatment) at either 44h post eCG or 11h post hCG.

Article Snippet: Sections were probed with primary antibodies against N-cadherin (BD Biosciences; Cat# 610920; 1:500); β-catenin (CST; Cat#8480; 1:500), E-cadherin (CST, Cat#14472; 1:500); a-tubulin (ThermoFisher; Cat#236-10501); Ki-67 (CST; Cat#9449; 1:1000) and Cleaved caspase 3 (CST; Cat#9661; 1:1000) diluted in 10% NGS and incubated O/N at at 4°C overnight in a humid chamber.

Techniques: Saline, Injection, Two Tailed Test, Staining, RNA Sequencing, Expressing, Quantitative RT-PCR, Control, Immunofluorescence

Journal: bioRxiv

Article Title: N-cadherin mechanosensing in ovarian follicles controls oocyte maturation and ovulation

doi: 10.1101/2023.10.06.561232

Figure Lengend Snippet: (a) qPCR analysis of relative mRNA expression of Cdh2, Areg, Ptgs2 and Cyp19a1 in ovaries of control ( Cdh2 Fl/+ ; Amhr2 Cre ) and granulosa-specific Cdh2 null mutants ( Cdh2 Fl/Fl ; Amhr2 Cre ), n=6 individual animals, *p<0.05. ( b-e ) Immunofluorescent analysis of N-cadherin protein in ovaries of control ( Cdh2 Fl/+ ; Amhr2 Cre ) and granulosa-specific Cdh2 null mutants ( Cdh2 Fl/Fl ; Amhr2 Cre ), showing mosaic depletion of N-cadherin in granulosa cells of mutant follicles. Arrows indicate mosaic regions with persistent N-cadherin. ( f ) Ovulation rate of 21d old mice with indicated control or granulosa-specific mutant genotypes. COCs in oviducts counted 16h after hCG injection. Graph represents mean ±SEM from N=4 and 7 animals respectively; **p<0.01 (unpaired two-tailed t-test). ( g ) Histology of ovaries by Haematoxylin & Eosin staining. Scale bar: 100µm.

Article Snippet: Sections were probed with primary antibodies against N-cadherin (BD Biosciences; Cat# 610920; 1:500); β-catenin (CST; Cat#8480; 1:500), E-cadherin (CST, Cat#14472; 1:500); a-tubulin (ThermoFisher; Cat#236-10501); Ki-67 (CST; Cat#9449; 1:1000) and Cleaved caspase 3 (CST; Cat#9661; 1:1000) diluted in 10% NGS and incubated O/N at at 4°C overnight in a humid chamber.

Techniques: Expressing, Control, Mutagenesis, Injection, Two Tailed Test, Staining

Journal: bioRxiv

Article Title: Synergistic Hypoxia and Apoptosis Conditioning Unleashes Superior Mesenchymal Stem Cells Efficacy in Acute Graft-versus-Host-Disease

doi: 10.1101/2024.04.11.588248

Figure Lengend Snippet: Effect of hypoxia-preconditioned MSCs (MSCs HYP , MSCs HYP→APO , MSCs HYP+APO ) on the proliferation of aGVHD patients derived T-cell. The bar graph represents (A) the percentage of CD3 + CFSE + proliferating T-cell (n=5). (B) the ratio of CD4 + /CD8 + T cell (n=5) in the direct co-culture of MSCs and T-cell. Data shown as Mean±S.D.; Statistical analysis: Tukey’s multiple comparisons test; **≤0.01; ****≤0.0001. Abbreviations: BM: Bone marrow; WJ: Wharton’s Jelly; MSCs: Mesenchymal Stem Cells; HYP: Hypoxia-preconditioned; APO: Apoptosis .

Article Snippet: CD3 + T-cell was isolated from PBMNCs by negative selection using a Pan T cell isolation kit, human (Miltenyi Biotec, USA) as per the manufacturer’s instructions.

Techniques: Derivative Assay, Co-Culture Assay

Journal: bioRxiv

Article Title: Synergistic Hypoxia and Apoptosis Conditioning Unleashes Superior Mesenchymal Stem Cells Efficacy in Acute Graft-versus-Host-Disease

doi: 10.1101/2024.04.11.588248

Figure Lengend Snippet: Dot plots showing the proliferation of CD3 + T-cell in the direct co-culture of aGVHD patients derived T-cell and (A) Untreated (Control). (B) BM-MSCs HYP . (C) BM-MSCs HYP→APO . (D) BM-MSCs HYP+APO . (E) WJ-MSCs HYP . (F) WJ-MSCs HYP→APO . (G) WJ-MSCs HYP+APO . Abbreviations: BM: Bone marrow; WJ: Wharton’s Jelly; MSCs: Mesenchymal Stem Cells; HYP: Hypoxia-preconditioned; APO: Apoptosis .

Article Snippet: CD3 + T-cell was isolated from PBMNCs by negative selection using a Pan T cell isolation kit, human (Miltenyi Biotec, USA) as per the manufacturer’s instructions.

Techniques: Co-Culture Assay, Derivative Assay, Control

Journal: bioRxiv

Article Title: Synergistic Hypoxia and Apoptosis Conditioning Unleashes Superior Mesenchymal Stem Cells Efficacy in Acute Graft-versus-Host-Disease

doi: 10.1101/2024.04.11.588248

Figure Lengend Snippet: Effect of hypoxia-preconditioned MSCs (MSCs HYP , MSCs HYP→APO , MSCs HYP+APO ) on the induction of Tregs and polarization of helper T cell from pro-inflammatory (Th1, Th17) to anti-inflammatory (Th2) phenotype. The bar graph represents (A) the induction of CD3 + CD4 + CD25 + FOXP3 + Tregs (n=5). (B) the ratio of Th1/Th2 (n=5). (C) Th1/Th17 ratio (n=5) in the direct co-culture of MSCs and T-cell. Data shown as Mean±S.D.; Statistical analysis: Tukey’s multiple comparisons test; ***≤0.001; ****≤0.0001. Abbreviations: BM: Bone marrow; WJ: Wharton’s Jelly; MSCs: Mesenchymal stem cells; HYP: Hypoxia-preconditioned; APO: Apoptosis, Tregs: Regulatory helper T-cell, Th: Helper T cell .

Article Snippet: CD3 + T-cell was isolated from PBMNCs by negative selection using a Pan T cell isolation kit, human (Miltenyi Biotec, USA) as per the manufacturer’s instructions.

Techniques: Co-Culture Assay

Journal: bioRxiv

Article Title: Synergistic Hypoxia and Apoptosis Conditioning Unleashes Superior Mesenchymal Stem Cells Efficacy in Acute Graft-versus-Host-Disease

doi: 10.1101/2024.04.11.588248

Figure Lengend Snippet: Dot plots showing the percentage of CD3 + CD4 + CD25 + FOXP3 + in the direct co-culture of aGVHD patients derived CD3 + T-cell and (A) Untreated (Control). (B) BM-MSCs HYP . (C) BM-MSCs HYP→APO . (D) BM-MSCs HYP+APO . (E) WJ-MSCs HYP . (F) WJ-MSCs HYP→APO . (G) WJ-MSCs HYP+APO . Abbreviations: BM: Bone marrow; WJ: Wharton’s Jelly; MSCs: Mesenchymal Stem Cells; HYP: Hypoxia-preconditioned; APO: Apoptosis .

Article Snippet: CD3 + T-cell was isolated from PBMNCs by negative selection using a Pan T cell isolation kit, human (Miltenyi Biotec, USA) as per the manufacturer’s instructions.

Techniques: Co-Culture Assay, Derivative Assay, Control