recombinant mouse thbs1  (Bio-Techne corporation)

Journal: Advanced Science

Article Title: Single‐Cell RNA Sequencing and Spatial Transcriptomics Reveal Pathogenesis of Meningeal Lymphatic Dysfunction after Experimental Subarachnoid Hemorrhage

doi: 10.1002/advs.202301428

Figure Lengend Snippet: Intercellular communication networks in and around mLVs. A) Spatial visualization of immune cell infiltration based on mLECs distribution. B) Outgoing and incoming signal patterns among all meningeal cells. C) THBS signaling pathway network among all cell types. D) Spatial visualization of THBS1‐CD47 L‐R pair distribution by stLearn. E) Volcano plots showing that THBS1 is one of the upregulated DEGs in the SAH group compared to the NPH group identified by mass spectrometry. F) Quantification of THBS1, THBS2, and THBS4 expression in CSF samples from SAH patients compared to those from NPH patients by ELISA assay, *** p < 0.001 versus NPH by paired two‐tailed Student's t ‐test. G) Representative confocal images of mLVs region of the negative control (NC) group and recombinant THBS1 protein treatment (rTHBS1) group. An enlarged view of TS and SSS is shown on the right in each group. Lyve1 (blue), Beads (green), and CD31 (red). Scale bar: 1000 µm. H) Flow cytometric analysis of mLECs percentage in NC group and rTHBS1 group, n = 4 per group, *** p < 0.001 versus NC by paired two‐tailed Student's t ‐test. I) Representative images of beads accumulation in dCLNs of the NC group and rTHBS1 group. Scale bar: 200 µm.

Article Snippet: For recombinant protein experiments, recombinant mice THBS1 protein (150 μg kg −1 , R&D Systems, 7859‐TH‐050, USA) or vehicle (PBS) were injected into cisterna magna CSF at a rate of 2 μL min −1 .

Techniques: Mass Spectrometry, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Negative Control, Recombinant

Journal: Advanced Science

Article Title: Single‐Cell RNA Sequencing and Spatial Transcriptomics Reveal Pathogenesis of Meningeal Lymphatic Dysfunction after Experimental Subarachnoid Hemorrhage

doi: 10.1002/advs.202301428

Figure Lengend Snippet: Cell trajectory analysis shows the evolution of mLECs. A) Labeling mLECs with real grouping information based on pseudotime trajectory map. B) Pseudotime trajectory map. C) Trajectory of representative genes in mLECs. D) Heatmap showing the top 30 pseudotime‐related genes. E) Representative two pseudo‐time‐related genes (S100 α 6 and Cldn5) based on q values. F,G) Increased expression of THBS1 and S100A6 are associated with poor prognosis, n = 48, * p < 0.05 by paired two‐tailed Student's t ‐test. H) Linear relationship between THBS1 and S100A6 expression, r = 0.3779, p = 0.0081 by Spearman's rank test. H) Representative confocal images of mLVs region of sham and SAH 24 h group. Enlarged view of selected region in the merged photo (yellow dotted box) are listed on the right in each group. Lyve1 (blue), S100 α 6 (green), and CD31 (red). Yellow arrows indicate where mLVs lie. Scale bar: 800 µm for the holistic view and 100 µm for the enlarged view.

Article Snippet: For recombinant protein experiments, recombinant mice THBS1 protein (150 μg kg −1 , R&D Systems, 7859‐TH‐050, USA) or vehicle (PBS) were injected into cisterna magna CSF at a rate of 2 μL min −1 .

Techniques: Labeling, Expressing, Two Tailed Test

Journal: Advanced Science

Article Title: Single‐Cell RNA Sequencing and Spatial Transcriptomics Reveal Pathogenesis of Meningeal Lymphatic Dysfunction after Experimental Subarachnoid Hemorrhage

doi: 10.1002/advs.202301428

Figure Lengend Snippet: Validation of THBS1 impacting mLVs function after SAH. SAH modeling and behavioral tests were performed 4 weeks after the AAV injection. A) Flow cytometric analysis of mLECs percentage in Sham, AAV‐control, and AAV‐THBS1 group at 72 h post‐SAH, n = 4 per group, ** p < 0.01, *** p < 0.001 by paired two‐tailed Student's t ‐test. B) Representative images and quantification of beads accumulation in dCLNs of Sham, AAV‐control, and AAV‐THBS1 group at 72 h post‐SAH, each data point represents an average of the 2 dCLNs from one individual mouse, n = 8 per group, ** p < 0.01, *** p < 0.001 by paired two‐tailed Student's t ‐test. Scale bar: 200 µm. C) Representative confocal images of mLVs region of Sham, AAV‐control, and AAV‐THBS1 group at 72 h post‐SAH. An enlarged view of TS and SSS is shown on the right in each group. Lyve1 (blue), Beads (green), and CD31 (red). Scale bar: 1000 µm. D) Modified Garcia test, E) time turn, F) time total, and G) wire hanging test at 72 h after SAH revealed AAV‐THBS1 delivery aggravated short‐term neurological function compared with Sham or AAV‐control group, n = 10–12 per group. * p < 0.05; ** p < 0.01, *** p < 0.001 by paired two‐tailed Student's t ‐test. H) Flow cytometric analysis of mLECs percentage in Sham, SAH‐WT, and THBS1‐KO group at 24 h post‐SAH, n = 4 per group, ** p < 0.01, *** p < 0.001 by paired two‐tailed Student's t ‐test. I) Representative images and quantification of beads accumulation in dCLNs in sham, SAH‐WT, and THBS‐KO group at 24 h post‐SAH, each data point represents an average of the 2 dCLNs from one individual mouse. n = 8 per group, ** p < 0.01 by paired two‐tailed Student's t ‐test. Scale bar: 200 µm. J) Representative confocal images of mLVs region of Sham, SAH‐WT, and THBS1‐KO group at 24 h post‐SAH. An enlarged view of TS and SSS is shown on the right in each group. Lyve1 (blue), Beads (green), and CD31 (red). Scale bar: 1000 µm. K) Modified Garcia test, L) time turn, M) time total, and N) wire hanging test at 24 h after SAH revealed THBS1 knockout improved short‐term neurological function compared with Sham or WT‐SAH group. n = 10–12 per group. * p < 0.05; ** p < 0.01, *** p < 0.001 by paired two‐tailed Student's t ‐test.

Article Snippet: For recombinant protein experiments, recombinant mice THBS1 protein (150 μg kg −1 , R&D Systems, 7859‐TH‐050, USA) or vehicle (PBS) were injected into cisterna magna CSF at a rate of 2 μL min −1 .

Techniques: Biomarker Discovery, Injection, Control, Two Tailed Test, Modification, Knock-Out

Journal: Advanced Science

Article Title: Single‐Cell RNA Sequencing and Spatial Transcriptomics Reveal Pathogenesis of Meningeal Lymphatic Dysfunction after Experimental Subarachnoid Hemorrhage

doi: 10.1002/advs.202301428

Figure Lengend Snippet: Disturbing THBS1‐CD47 interaction promoted mLVs restoration via inhibiting STAT3/BCL2‐mediated apoptosis in mLECs. A) Flow cytometric analysis of mLECs percentage in sham, SAH + igG, SAH + anti‐CD47, and SAH + anti‐THBS1 group at 24 h post‐SAH, n = 4 per group, *** p < 0.001 by paired two‐tailed Student's t ‐test. B) Representative images and quantification of beads accumulation in dCLNs of Sham, SAH + igG, SAH + anti‐CD47, and SAH + anti‐THBS1 group at 24 h post‐SAH, each data point represents an average of the 2 dCLNs from one individual mouse, n = 8 per group, *** p < 0.001 by paired two‐tailed Student's t ‐test. Scale bar: 200 µm. C) Representative confocal images of mLVs region of Sham, SAH + igG, SAH + anti‐CD47, and SAH + anti‐THBS1 group at 24 h post‐SAH. An enlarged view of TS and SSS is shown on the right in each group. Lyve1 (blue), Beads (green), and CD31 (red). Scale bar: 1000 µm. D) Modified Garcia test, E) time turn, F) time total, and G) wire hanging test at 24 h after SAH revealed anti‐CD47 and anti‐THBS1 therapy improved short‐term neurological function compared with Sham or SAH + igG group. n = 10–12 per group. * p < 0.05; ** p < 0.01, *** p < 0.001 by paired two‐tailed Student's t ‐test. H) GSEA showed apoptosis pathway was activated in mLECs after SAH. I) Representative immunoblot images showing effects of rTHBS1 (100 ng mL −1 ) treatment on pSTAT3 and Bcl‐2 inhibition in primary mLECs. J–M) Representative immunoblot images of STAT3, pSTAT3, Bax, and Bcl‐2 in different groups.

Article Snippet: For recombinant protein experiments, recombinant mice THBS1 protein (150 μg kg −1 , R&D Systems, 7859‐TH‐050, USA) or vehicle (PBS) were injected into cisterna magna CSF at a rate of 2 μL min −1 .

Techniques: Two Tailed Test, Modification, Western Blot, Inhibition