Journal: Advanced Science

Article Title: Sirtuin1 Suppresses Calcium Oxalate Nephropathy via Inhibition of Renal Proximal Tubular Cell Ferroptosis Through PGC‐1α‐mediated Transcriptional Coactivation

doi: 10.1002/advs.202408945

Figure Lengend Snippet: Sirt1 inhibited CaOx‐induced ferroptosis in tubular epithelium. A) Sirt1 activation and overexpression restored the cell viability of COM‐treated HK‐2 cells evaluated by CCK‐8. B, C) Lipid peroxidation in HK‐2 cells was determined by BODIPY C11 staining and flow cytometry. D) Sirt1 activation and overexpression reduced the level of MDA in COM‐treated HK‐2 cells. Immunoblotting E) and qPCR F) revealed the expression of ferroptosis associated markers in HK‐2 cells. In vivo, mice were pretreated with SRT1720/EX527 and then induced to establish a model of CaOx nephrocalcinosis. Immunoblotting G) and qPCR H) revealed the expression of ferroptosis associated markers in HK‐2 cells. The deposition of CaOx was observed under a polarized light microscopy I) and evaluated by Pizzolato staining J). K) PAS staining was used for the scoring of tubular injury. The blue arrows highlight damaged renal tubules. L) IHC staining was performed to test the level of GPX4 and 4HNE in mouse kidney. M) The changes of mitochondrial morphology in tubular epithelium were observed by TEM. The red arrows indicate the morphologically abnormal mitochondria, characterized by mitochondrial shrinkage and disorganized cristae. N, O) The serum levels of creatinine and BUN were detected to assess kidney function. P) MDA in tissues of mice kidney was quantified. Q) The expression of KIM‐1 mRNA in tissues of mice kidney was determined by qPCR. Data were presented as mean ± SD, n = 3 for in vitro experiments and n = 6 for in vivo experiments. P value was determined by one‐way (C, D, F, H, N‐Q) or two‐way (A) ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Renal tubular epithelium‐specific Sirt1 knockout (Sirt1cKO) mice were obtained by crossing Cdh16‐Cre males (T007046, GemPharmatech Inc., China) with Sirt1 fl/fl females (T006657, GemPharmatech Inc.) on a C57BL/6J background.

Techniques: Activation Assay, Over Expression, CCK-8 Assay, Staining, Flow Cytometry, Western Blot, Expressing, In Vivo, Light Microscopy, Immunohistochemistry, In Vitro

Journal: Advanced Science

Article Title: Sirtuin1 Suppresses Calcium Oxalate Nephropathy via Inhibition of Renal Proximal Tubular Cell Ferroptosis Through PGC‐1α‐mediated Transcriptional Coactivation

doi: 10.1002/advs.202408945

Figure Lengend Snippet: Sirt1 inhibited CaOx‐induced ferroptosis in tubular epithelium dependent on GPX4.HK‐2 cells were treated with increasing concentrations of erastin, along with 5 µM SRT1720 or 1 µM Fer for 24h. A) Cell viability of HK‐2 cells was evaluated by CCK‐8. B, C) Lipid peroxidation in HK‐2 cells was determined by BODIPY C11 staining and flow cytometry. D) Level of MDA in HK‐2 cells treated with erastin, along with SRT1720 or Fer for 24 h. (E) HK‐2 cells were treated with increasing concentrations of RSL3, along with 5 µM SRT1720 or 1 µM Fer for 24 h. Cell viability of HK‐2 cells was evaluated by CCK‐8. F, G) Lipid peroxidation in HK‐2 cells was determined by BODIPY C11 staining and flow cytometry. H) Level of MDA in HK‐2 cells treated with erastin, along with SRT1720 or Fer for 24 h. I) Cell viability of Sh ctrl and GPX4 sh HK‐2 cells treated with increasing concentrations of COM and SRT1720 was evaluated by CCK‐8. J, K) Lipid peroxidation in Sh ctrl and GPX4 sh HK‐2 cells was determined by BODIPY C11 staining and flow cytometry. L) Level of MDA in Sh ctrl and GPX4 sh HK‐2 cells treated with increasing concentrations of COM and SRT1720 for 24 h. Data were presented as mean ± SD, n = 3, and P value was determined by one‐way (C, D, G, H, K, L) or two‐way (A, E, I) ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Renal tubular epithelium‐specific Sirt1 knockout (Sirt1cKO) mice were obtained by crossing Cdh16‐Cre males (T007046, GemPharmatech Inc., China) with Sirt1 fl/fl females (T006657, GemPharmatech Inc.) on a C57BL/6J background.

Techniques: CCK-8 Assay, Staining, Flow Cytometry

Journal: Advanced Science

Article Title: Sirtuin1 Suppresses Calcium Oxalate Nephropathy via Inhibition of Renal Proximal Tubular Cell Ferroptosis Through PGC‐1α‐mediated Transcriptional Coactivation

doi: 10.1002/advs.202408945

Figure Lengend Snippet: Sirt1 inhibited ferroptosis in tubular epithelium through the PGC‐1α/GPX4 pathway.A, B) Sirt1 activation and overexpression increased the protein and mRNA expression of PGC‐1α. C, D) The protein and mRNA levels of GPX4 in Sh ctrl and PGC‐1α sh HK‐2 cells treated by ZLN005. E) Cell viability of Sh ctrl and PGC‐1α sh HK‐2 cells treated with increasing concentrations of COM and SRT1720 was evaluated by CCK‐8. F, G) Lipid peroxidation in Sh ctrl and PGC‐1α sh HK‐2 cells was determined by BODIPY C11 staining and flow cytometry. Immunoblotting H) and qPCR I) revealed the expression of ferroptosis associated markers in Sh ctrl and PGC‐1α sh HK‐2 cells treated by COM and SRT1720. J) Cell viability of Sh ctrl and PGC‐1α sh HK‐2 cells treated with increasing concentrations of erastin and SRT1720 was evaluated by CCK‐8. K, L) Lipid peroxidation in Sh ctrl and PGC‐1α sh HK‐2 cells was determined by BODIPY C11 staining and flow cytometry. Immunoblotting M) and qPCR N) revealed the expression of ferroptosis associated markers in Sh ctrl and PGC‐1α sh HK‐2 cells treated by erastin and SRT1720. Data were presented as mean ± SD, n = 3, and P value was determined by one‐way (B, D, G, I, L, N) or two‐way (E, J) ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Renal tubular epithelium‐specific Sirt1 knockout (Sirt1cKO) mice were obtained by crossing Cdh16‐Cre males (T007046, GemPharmatech Inc., China) with Sirt1 fl/fl females (T006657, GemPharmatech Inc.) on a C57BL/6J background.

Techniques: Activation Assay, Over Expression, Expressing, CCK-8 Assay, Staining, Flow Cytometry, Western Blot

Journal: Advanced Science

Article Title: Sirtuin1 Suppresses Calcium Oxalate Nephropathy via Inhibition of Renal Proximal Tubular Cell Ferroptosis Through PGC‐1α‐mediated Transcriptional Coactivation

doi: 10.1002/advs.202408945

Figure Lengend Snippet: Sirt1 inhibited CaOx‐induced ferroptosis, crystal deposition and kidney injury through PGC‐1α/NRF2 signaling.A) The conditional knockout of Sirt1 in tubular epithelium in mouse kidney. B) Schematic of establishment of CaOx nephrocalcinosis model and treatment of ZLN005 and TBHQ. The deposition of CaOx was observed under a polarized light microscopy C) and evaluated by Pizzolato staining D). E) PAS staining was used for the scoring of tubular injury. The blue arrows highlight damaged renal tubules. F) IHC staining was performed to test the level of GPX4 and 4HNE in mouse kidney. G) The levels of renal ROS were detected by DHE staining. H, I) The serum levels of creatinine and BUN were detected to assess kidney function. J) MDA in tissues of mice kidney was quantified. Data were presented as mean ± SD, n = 6, and P value was determined by one‐way ANOVA (H‐J). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Renal tubular epithelium‐specific Sirt1 knockout (Sirt1cKO) mice were obtained by crossing Cdh16‐Cre males (T007046, GemPharmatech Inc., China) with Sirt1 fl/fl females (T006657, GemPharmatech Inc.) on a C57BL/6J background.

Techniques: Knock-Out, Light Microscopy, Staining, Immunohistochemistry

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: 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