Journal: International Wound Journal

Article Title: Negative‐Pressure Induces Epithelial‐Mesenchymal Transition via Thrombospondin‐1 Upregulation in Intact Diabetic Skin

doi: 10.1111/iwj.70859

Figure Lengend Snippet: EMT‐related protein and marker expression induced by negative‐pressure in vitro. (a) Enzyme‐linked immunosorbent assay (ELISA) of culture supernatants and cell lysates revealed significantly increased protein levels of thrombospondin‐1 (THBS1) ( p = 0.008), transforming growth factor‐beta 1 (TGF‐β1) ( p = 0.008), plasminogen activator inhibitor‐1 (PAI‐1) ( p = 0.001) and hypoxia‐inducible factor‐1 alpha (HIF‐1α) ( p = 0.022) in keratinocytes cultured under negative‐pressure (NP) compared with ambient pressure (AP). (b) Epithelial‐mesenchymal transition (EMT) marker expression showed a significant decrease in E‐cadherin ( p = 0.031) and an increase in alpha‐smooth muscle actin (αSMA) ( p = 0.031) in the NP group; vimentin expression also tended to increase ( p = 0.051). n = 12 for each group. Values are presented as means ± standard deviations. Statistical significance is indicated as * p < 0.05 and ** p < 0.01. αSMA: Alpha‐smooth muscle actin, AP: ambient pressure, ELISA: enzyme‐linked immunosorbent assay, EMT: epithelial‐mesenchymal transition, HIF‐1α: hypoxia‐inducible factor‐1 alpha, NP: negative‐pressure, PAI‐1: plasminogen activator inhibitor‐1, TGF‐β1: transforming growth factor‐beta 1, THBS1: thrombospondin‐1.

Article Snippet: THBS1 , Hs00962908_ml , THBS1.

Techniques: Marker, Expressing, In Vitro, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: International Wound Journal

Article Title: Negative‐Pressure Induces Epithelial‐Mesenchymal Transition via Thrombospondin‐1 Upregulation in Intact Diabetic Skin

doi: 10.1111/iwj.70859

Figure Lengend Snippet: Gene expression in PSVK1 keratinocytes exposed to negative‐pressure and THBS1 inhibition. Real‐time polymerase chain reaction (PCR) was performed on cultured human keratinocyte PSVK1 cells exposed to ambient pressure (AP) or negative‐pressure (NP), with or without treatment with Leucine‐Serine‐Lysine‐Leucine (LSKL), a thrombospondin‐1 (THBS1) inhibitor or dimethyl sulfoxide (DMSO), a vehicle control. TaqMan assays revealed that THBS1 expression was reduced under NP when treated with LSKL. Transforming growth factor‐beta 1 ( TGFB1 ) expression also decreased with LSKL under AP. Cadherin 1 ( CDH1 ), which encodes E‐cadherin, was downregulated under NP regardless of treatment. Vimentin ( VIM ) expression increased under NP with vehicle but showed a decreasing trend with LSKL. Actin alpha 2 ( ACTA2 ), encoding αSMA, was consistently downregulated by LSKL treatment under both pressure conditions. Data represent eight replicates per group and were analysed using two‐way analysis of variance with Tukey's range test. Significance is indicated as * p < 0.05, ** p < 0.01 and *** p < 0.001. ACTA2: actin alpha 2, αSMA: alpha‐smooth muscle actin, CDH1: Cadherin 1, DMSO: dimethyl sulfoxide, LSKL: Leucine‐Serine‐Lysine‐Leucine, PCR: polymerase chain reaction, THBS1: thrombospondin‐1, TGF‐β1: transforming growth factor‐beta 1, VIM: vimentin.

Article Snippet: THBS1 , Hs00962908_ml , THBS1.

Techniques: Gene Expression, Inhibition, Real-time Polymerase Chain Reaction, Cell Culture, Control, Expressing, Polymerase Chain Reaction

Journal: International Wound Journal

Article Title: Negative‐Pressure Induces Epithelial‐Mesenchymal Transition via Thrombospondin‐1 Upregulation in Intact Diabetic Skin

doi: 10.1111/iwj.70859

Figure Lengend Snippet: Histological and immunohistochemical changes in NP‐treated diabetic mouse skin. (a) Immunohistochemistry (IHC) staining was performed to evaluate the epithelial‐mesenchymal transition (EMT)‐related marker thrombospondin‐1 (THBS1). THBS1‐positive pixels in the epidermis were quantified. THBS1 positivity was significantly higher in the negative‐pressure (NP) group compared with the other two groups ( p < 0.001). (b) Skin thickness was assessed in haematoxylin and eosin (HE)‐stained sections. Both epidermal and dermal thickness were significantly greater in the NP group than in the other two groups ( p < 0.001). (c) IHC staining for hypoxia‐inducible factor‐1 alpha (HIF‐1α), plasminogen activator inhibitor‐1 (PAI‐1), and fibrinogen was performed to evaluate tissue hypoxia and microthrombosis induced by NP. (c1) PAI‐1 positivity in the epidermis was significantly higher in the NP group ( p < 0.001). (c2) The number of HIF‐1α positive cells per mm 2 in the epidermis was significantly increased in the NP group ( p < 0.001). (c3) The number of fibrinogen‐positive vessels per mm 2 in the dermis was significantly higher in the NP group ( p < 0.001). Arrowheads indicate fibrinogen‐positive vessels. (d) Angiogenesis was evaluated using IHC for CD31. The number of CD31‐positive vessels per mm 2 in the dermis was significantly higher in the NP group ( p < 0.001). Arrowheads indicate CD31‐positive vessels. N = 6 each. Values are presented as means ± standard deviations. Statistical significance is indicated as *** p < 0.001. Bar: 50 μm. AP: ambient pressure, ELISA: enzyme‐linked immunosorbent assay, EMT: epithelial‐mesenchymal transition, HE: haematoxylin and eosin, HIF‐1α: hypoxia‐inducible factor‐1 alpha, IHC: immunohistochemistry, NP: negative‐pressure, PAI‐1: plasminogen activator inhibitor‐1, THBS1: thrombospondin‐1, TGF‐β1: transforming growth factor‐beta 1.

Article Snippet: THBS1 , Hs00962908_ml , THBS1.

Techniques: Immunohistochemical staining, Immunohistochemistry, Marker, Staining, Enzyme-linked Immunosorbent Assay

Journal: International Wound Journal

Article Title: Negative‐Pressure Induces Epithelial‐Mesenchymal Transition via Thrombospondin‐1 Upregulation in Intact Diabetic Skin

doi: 10.1111/iwj.70859

Figure Lengend Snippet: Quantitative analysis of EMT‐ and angiogenesis‐related proteins in NP‐treated mouse skin. Enzyme‐linked immunosorbent assay (ELISA) of skin tissue samples revealed significantly increased levels of thrombospondin‐1 (THBS1), transforming growth factor‐beta 1 (TGF‐β1), plasminogen activator inhibitor‐1 (PAI‐1), hypoxia‐inducible factor‐1 alpha (HIF‐1α) and vascular endothelial growth factor (VEGF) in the negative‐pressure (NP) group compared with the control and ambient pressure (AP) groups. THBS1: p = 0.012 versus control, p = 0.011 versus AP; TGF‐β1: p = 0.008 versus control, p = 0.006 versus AP; PAI‐1: p < 0.001 versus both; HIF‐1α: p = 0.004 versus control, p = 0.001 versus AP; VEGF: p < 0.001 versus both. N = 6 per group. Values are presented as means ± standard deviations. Statistical significance is indicated as * p < 0.05, ** p < 0.01 and *** p < 0.001. AP: ambient pressure, ELISA: enzyme‐linked immunosorbent assay, EMT: epithelial‐mesenchymal transition, HIF‐1α: hypoxia‐inducible factor‐1 alpha, NP: negative‐pressure, PAI‐1: plasminogen activator inhibitor‐1, THBS1: thrombospondin‐1, TGF‐β1: transforming growth factor‐beta 1, VEGF: vascular endothelial growth factor.

Article Snippet: THBS1 , Hs00962908_ml , THBS1.

Techniques: Enzyme-linked Immunosorbent Assay, Control

Journal: Neuron

Article Title: Thrombospondin-1 Mediates Axon Regeneration in Retinal Ganglion Cells

doi: 10.1016/j.neuron.2019.05.044

Figure Lengend Snippet: (A and B) Images of optic nerve sections showing CTB-labeled axons (grey) in C57BL/6J mice injected with either (A) AAV-THBS1 or (B) AAV-GFP. Asterisks, lesion site. Scale bars, 100 μm.

Article Snippet: To generate the pAAV.GFAP.SV40.Thbs1-HA.SV40(polyA) plasmid, the 377 bp CMV promoter coding sequence was deleted from pAAV.CMV.SV40.THBS1-HA.SV40(polyA) plasmid by digestion with AvrII and BspEI restriction enzymes. pAAV.GFAP.EGFP plasmid (Addgene #50473) was used as template to PCR amplify short GFAP promoter sequence using the following oligonucleotides: GFAP-F and GFAP-R.

Techniques: Labeling, Injection

Journal: Neuron

Article Title: Thrombospondin-1 Mediates Axon Regeneration in Retinal Ganglion Cells

doi: 10.1016/j.neuron.2019.05.044

Figure Lengend Snippet: (A) A schematic of THBS1 mutants investigated. All constructs contain the N-terminal signal peptide and have a C-terminal HA tag. Laminin G domain (LamG), oligomerization coiled coil (CC) domain, von Willebrand complex like domain (vWC), thrombospondin type 1 repeat domain (TSR1), epidermal growth factor-like repeat domains (EGF), type 3 repeat domain (TSR3), and the thrombospondin C-terminal domain (CTD). THBS4 is shown for comparison to THBS1.

Article Snippet: To generate the pAAV.GFAP.SV40.Thbs1-HA.SV40(polyA) plasmid, the 377 bp CMV promoter coding sequence was deleted from pAAV.CMV.SV40.THBS1-HA.SV40(polyA) plasmid by digestion with AvrII and BspEI restriction enzymes. pAAV.GFAP.EGFP plasmid (Addgene #50473) was used as template to PCR amplify short GFAP promoter sequence using the following oligonucleotides: GFAP-F and GFAP-R.

Techniques: Construct

Journal: Neuron

Article Title: Thrombospondin-1 Mediates Axon Regeneration in Retinal Ganglion Cells

doi: 10.1016/j.neuron.2019.05.044

Figure Lengend Snippet: (A) Images of optic nerve section showing GFP-labeled axons (green) from HB9:GFP;Bax−/− mice and CTB (magenta) following injection with AAV-THBS1 and optic nerve crush. Asterisks, lesion site.

Article Snippet: To generate the pAAV.GFAP.SV40.Thbs1-HA.SV40(polyA) plasmid, the 377 bp CMV promoter coding sequence was deleted from pAAV.CMV.SV40.THBS1-HA.SV40(polyA) plasmid by digestion with AvrII and BspEI restriction enzymes. pAAV.GFAP.EGFP plasmid (Addgene #50473) was used as template to PCR amplify short GFAP promoter sequence using the following oligonucleotides: GFAP-F and GFAP-R.

Techniques: Labeling, Injection

Journal: Neuron

Article Title: Thrombospondin-1 Mediates Axon Regeneration in Retinal Ganglion Cells

doi: 10.1016/j.neuron.2019.05.044

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: To generate the pAAV.GFAP.SV40.Thbs1-HA.SV40(polyA) plasmid, the 377 bp CMV promoter coding sequence was deleted from pAAV.CMV.SV40.THBS1-HA.SV40(polyA) plasmid by digestion with AvrII and BspEI restriction enzymes. pAAV.GFAP.EGFP plasmid (Addgene #50473) was used as template to PCR amplify short GFAP promoter sequence using the following oligonucleotides: GFAP-F and GFAP-R.

Techniques: shRNA, Recombinant, Multiplex Assay, Clone Assay, Software

Journal: International Journal of Molecular Sciences

Article Title: Elevated Levels of Active GSK3β in the Blood of Patients with Myotonic Dystrophy Type 1 Correlate with Muscle Weakness

doi: 10.3390/ijms262110760

Figure Lengend Snippet: Levels of active GSK3β are increased in PBMCs from patients with adult-onset DM1 relative to unaffected control patients. ( A ) The scheme shows the preparation of PBMC proteins for Western blot analyses. The whole blood samples were separated by centrifugation in the gradient of Biocoll. The buffy coats were collected, and PBMCs were spun down. The cell pellets were resuspended in SDS-loading buffer, boiled for 30 min, and analyzed by Western blot assay as described in the Methods section. ( B ) Western blot analyses of proteins from PBMCs from control and adult-onset DM1 patients. Control samples 1–3 are familial controls not affected by DM1 (a 56-year-old female, a 49-year-old male and 55-year-old male, respectively). Control samples 4–7 are not familial participants (a 59-year-old female, a 35-year-old female, a 66-year-old male and 28-year-old female). The labels for DM1 samples include the length of CTG repeats. The DM1–68, DM1–74, DM1–100, DM1–131, DM1–163, DM1–>200 and DM1–~500 are samples from participants with adult-onset DM1 (females of 48, 43, 59, 31, 40, 29 and 40 years, respectively). Coomassie blue staining of the proteins extracted from PBMCs is shown on the top. Active GSK3β, total GSK3β and THBS1 were measured. Ph to tot: numbers below the total GSK3β image represent the ratios of the active GSK3β signals to total GSK3β in the same DM1 blood samples. ( C ) Bar graphs of the active GSK3β signals shown in ( B ) relative to Coomassie signals. Standard deviations (SDs) calculated from three repeats are shown. ( D ) Weighted regression results of active GSK3β levels on CTG repeat number, using data from 1B. For each DM1 subject, the dependent variable is the mean active GSK3β, and the weight is the inverse of the variance. Predicted GSK3β is shown with shaded 95% confidence bands. Standard deviation bars are calculated from three replicates. Colors denote the patients. ( E ) Western blot analysis of additional unrelated control blood samples vs. DM1 samples with antibodies to active GSK3β. C8, C10, C11, C13 and C14 are females of 41, 28, 38 and 52 years of age, respectively. C9 and C12 are males of 69 and 62 years. DM1 samples from patients with 163 and >200 CTG repeats analyzed in ( B ) are used as positive controls. Coomassie staining of the proteins extracted from PBMCs is shown on the bottom. ( F ) Using the same approach applied to GSK3β, a weighted regression was performed with THBS1 levels from 1B as the dependent variable and CTG repeat number as a predictor. For control samples, the maximum limit of 50 CTG repeats was applied because the exact number of repeats in these unaffected controls is not known. Bars indicate THBS1 standard deviations from three repeats. Predicted THBS1 is shown with shaded 95% confidence bands.

Article Snippet: To examine GSK3β, THBS1, TGFβ and hnRNP-A3 in the human PBMCs; 10 μg of total proteins from PBMCs were separated by the 4–20% polyacrylamide gel electrophoresis; proteins were transferred onto a nitro cellulose membrane and probed with primary antibodies to phospho-GSK3β-Y216 (EpigenTek, New York, NY, USA), total GSK3β and β-actin (Santa Cruz Biotechnology Inc., Dallas, TX, USA), THBS1 (Proteintech Group, Inc., Rosemont, IL, USA), TGFβ (MilliporeSigma, Burlington, VT, USA) and hnRNP-A3 (ThermoFisher, Dallas, TX, USA) according to the manufactures’ suggestions overnight.

Techniques: Control, Western Blot, Centrifugation, Staining, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Elevated Levels of Active GSK3β in the Blood of Patients with Myotonic Dystrophy Type 1 Correlate with Muscle Weakness

doi: 10.3390/ijms262110760

Figure Lengend Snippet: Correlation of the average levels of active GSK3β in blood with the muscle performance of DM1 patients with different severity of muscle disease. ( A ) Comparison of the average GSK3β levels in PBMCs from adult-onset DM1 patients with 68–100 CTG repeats without myotonia (n = 3) and in patients with 131–500 CTG repeats with myotonia and muscle weakness (n = 4). Western blot data and signal intensity analyses (using Image J2) are shown in B,C. A group of DM1 patients with mild muscle disease includes DM1 patients: DM1–68, DM1–74 and DM–100. A group of DM1 patients with moderate muscle weakness includes DM1–131, DM1–163, DM1 > 200 and DM1 ~ 500. The values of grip strength ( B , C ) and ankle strength ( D , E ) were compared in adult-onset DM1 patients with mild (n = 3) and moderate (n = 4) skeletal muscle weakness. The skeletal muscle outcomes for each patient with DM1 are shown in A,B. ( F ) Comparison of the blood levels of THBS1 in adult-onset DM1 patients with mild and moderate skeletal muscle disease. Western blot analyses of THBS1 in DM1 blood samples are shown in B. Standard deviations and the number of DM1 patients is shown. * and ** are p values < 0.05 and <0.01 for the DM1 patients with mild vs. moderate muscle weakness.

Article Snippet: To examine GSK3β, THBS1, TGFβ and hnRNP-A3 in the human PBMCs; 10 μg of total proteins from PBMCs were separated by the 4–20% polyacrylamide gel electrophoresis; proteins were transferred onto a nitro cellulose membrane and probed with primary antibodies to phospho-GSK3β-Y216 (EpigenTek, New York, NY, USA), total GSK3β and β-actin (Santa Cruz Biotechnology Inc., Dallas, TX, USA), THBS1 (Proteintech Group, Inc., Rosemont, IL, USA), TGFβ (MilliporeSigma, Burlington, VT, USA) and hnRNP-A3 (ThermoFisher, Dallas, TX, USA) according to the manufactures’ suggestions overnight.

Techniques: Comparison, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Elevated Levels of Active GSK3β in the Blood of Patients with Myotonic Dystrophy Type 1 Correlate with Muscle Weakness

doi: 10.3390/ijms262110760

Figure Lengend Snippet: The levels of active GSK3β are increased in PBMCs of patients with juvenile and congenital DM1 correlating with muscle weakness. ( A ) Western blot analyses of the active GSK3β and THBS1 in PBMCs from the familial control patients (C1 and C2 are a 56-year-old female and a 49-year-old male, respectively), patients with JDM1 (17- and 19-year-old females) and CDM1 (CDM1-1–4 are an 18-year-old female, a 16-year-old male, a 5-year-old female and a 5-month-old female, respectively). Coomassie staining of the PBMC proteins is shown below the Western blot images. ( B ) The average active GSK3β levels in the PBMCs from control, JDM1 and CDM1 patients were adjusted to the Coomassie-stained proteins shown in the box in ( A ). * and *** are p values < 0.05 and <0.001 for the average GSK3β signal in JDM1 compared to control and for the average GSK3β in CDM1 patients vs. control. ( C ) Comparison of the mean length of CTG repeat expansions in JDM1 and CDM1 patients. ** is p value < 0.01 for JDM1 vs. CDM1. ( D ) Western blot analysis of active GSK3β in pediatric and adolescent control blood samples and CDM1 samples. Coomassie staining is shown on the bottom. The average values of ankle dorsiflexion ( E , F ), grip strength ( G , H ) and manual strength ( I , J ) in patients with JDM1 and CDM1 are shown. The number of patients per group is also shown. * indicates a p value < 0.05 for JDM1 vs. CDM1. While GSK3β levels were evaluated in blood samples from four patients with CDM1, the muscle performance was analyzed in two patients with CDM1 aged 16 and 18. No muscle performance data were collected in two CDM1 patients due to early age (0.5 and 5 years).

Article Snippet: To examine GSK3β, THBS1, TGFβ and hnRNP-A3 in the human PBMCs; 10 μg of total proteins from PBMCs were separated by the 4–20% polyacrylamide gel electrophoresis; proteins were transferred onto a nitro cellulose membrane and probed with primary antibodies to phospho-GSK3β-Y216 (EpigenTek, New York, NY, USA), total GSK3β and β-actin (Santa Cruz Biotechnology Inc., Dallas, TX, USA), THBS1 (Proteintech Group, Inc., Rosemont, IL, USA), TGFβ (MilliporeSigma, Burlington, VT, USA) and hnRNP-A3 (ThermoFisher, Dallas, TX, USA) according to the manufactures’ suggestions overnight.

Techniques: Western Blot, Control, Staining, Comparison

Journal: Advanced Science

Article Title: Genome‐Wide CRISPR Screen Identifies a microRNA Orchestrating Pleiotropic Resistance to Targeted Therapy and T Cell Immunity in Melanoma

doi: 10.1002/advs.202515158

Figure Lengend Snippet: miR‐18a regulates the TGF‐β signaling pathway to confer CD8 + T cell tolerance by targeting THBS1 . (A) Volcano plot showing differential expression of TGF‐β pathway genes, with miR‐18a‐5p targets (diamonds) and miR‐18a‐3p targets (triangles) highlighted and THBS1 labeled. (B) Western blot of THBS1 in A375‐Cas9 (control) and miR‐18a KO cells. (C) Dual‐luciferase reporter assay showing miR‐18a‐5p mimics suppress WT THBS1 3′UTR activity but not MUT 3′UTR. NC: scrambled mimic control. (D) Western blot confirming siRNA‐mediated THBS1 knockdown (438, 1955, and 2095) in miR‐18a KO A375 cells. Control: scrambled RNA. (E) Cell proliferation of A375‐Cas9 (control) and miR‐18a KO cells co‐cultured with CD8 + T cells at various T:E ratios with scrambled RNA (NC) or si THBS1 (2095). (F) Schematic of C57BL/6 murine tumor model with miR‐18a‐5p mimic treatment. Mice were inoculated with miR‐18a KO YUMM1.7 cells and subsequently injected with 20 µL of scrambled mimic control or miR‐18a‐5p mimics. (G) Representative tumor images and tumor growth curves in miR‐18a‐5p mimic‐treated versus control mice. (H) Single‐cell UMAP of tumor tissues from control and miR‐18a‐5p mimic‐treated mice. (I) Hallmark pathway enrichment of DEGs (miR‐18a‐5p mimic versus control), showing TGF‐β suppression. FDTs: Fibroblast‐Derived Tumor cells; EDTs: Epithelial‐Derived Tumor cells. (J) Signaling interactions between FDTs and CD8 + T cells in control versus miR‐18a‐5p mimic‐treated mice. (K) Thbs1 expression across cell types in control and miR‐18a‐5p mimic‐treated mice. (L) Cell proliferation of A375‐Cas9 (control) and miR‐18a KO A375 cells with/without CD8 + T cell and CD47 inhibitor (magrolimab; isotype IgG control). (M, N) Representative IHC images (M) and quantification (N) of Ki67, Cd47, and Thbs1 in murine tumor tissues. Data represent means ± SD; n = 3 biologically independent samples for panels C and E, n = 6 for panel L, and n = 7 for panels G and N. Two‐way ANOVA followed by Tukey's post hoc test is used to calculate P ‐values in panel G and unpaired two‐tailed Student's t ‐test is used to calculate P ‐values in panels C, E, L, and N: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

Article Snippet: Primary antibodies used were as follows: phospho‐MEK (Cell Signaling Technology, 3958S, RRID: AB_2138014; 1:1,000), total MEK (Cell Signaling Technology, 4694S, RRID: AB_10695868; 1:2,000), phospho‐AKT (Cell Signaling Technology, 4060S, RRID: AB_2315049; 1:1,000), total AKT (Cell Signaling Technology, 2920S, RRID: AB_1147620; 1:2,000), phospho‐ERK (Cell Signaling Technology, 4377S, RRID: AB_331775; 1:1,000), total ERK (Cell Signaling Technology, 4695S, RRID: AB_390779; 1:2,000), AJUBA (ABclonal, A22039, RRID: AB_3719524; 1:2,000), CD47 (ABclonal, A11382, RRID: AB_2861554; 1:2,000), THBS1 (ABclonal, A2125, RRID: AB_2764144; 1:2,000), and hnRNP A1 (ABclonal, A11564, RRID: AB_2861599; 1:2,000). β‐actin (ABclonal, AC026, RRID: AB_2768234; 1:10,000) or GAPDH (ABclonal, AC001, RRID: AB_2619673; 1:10,000) served as loading controls.

Techniques: Quantitative Proteomics, Labeling, Western Blot, Control, Luciferase, Reporter Assay, Activity Assay, Knockdown, Cell Culture, Injection, Single Cell, Derivative Assay, Expressing, Two Tailed Test

Journal: Methods and Protocols

Article Title: Efficient Ex Vivo Screening of Agents Targeting Thrombospondin1-Induced Vascular Dysfunction Using a Digital Multiwire Myograph System

doi: 10.3390/mps4040074

Figure Lengend Snippet: Native thrombospondin 1 (TSP1) from different sources inhibited endotheliumdependent vasorelaxation with different efficacies. ( A ) Representative force vs. time traces recordings of isolated murine thoracic aorta responses to phenylephrine followed acetylcholine stimulations in the presence of Pierce TM —TSP1 or vehicle control, i.e., Krebs buffer (PE: phenylephrine; Ach: acetylcholine; the numbers marked in the recording are the logarithm value of concentrations of the stimulants). Concentration response curves of isolated mouse thoracic aorta exposed to Pierce TM —TSP1 ( B ) or Athens—TSP1 ( C ) at 2.2, 4.4, or 6.6 nM. Pierce TM —TSP1 induced impairment of vasodilation in a slightly different pattern than Athens—TSP1 (*, p < 0.05, TSP1 vs. control; ***, p < 0.0001, TSP1 vs. control). ( D ) The same concentration TSP1, e.g., 4.4 nM, from three sources inhibited vasorelaxation, two of which produced greater suppression of vasorelaxation induced by acetylcholine 1 × 10 −7 and 3 × 10 −7 M (*, p < 0.05, Athens—TSP1 vs. control; ***, p < 0.0001, Pierce TM —TSP1 or Novus—TSP1 vs. control). ( E ) An equivalent inhibition of vasorelaxation was reached when Novus—TSP1 or Athens—TSP1 at 2.2 nM and Pierce TM —TSP1 at 4.4 nM was applied (***, p < 0.0001, TSP1 vs. control).

Article Snippet: Therefore, it was essential to examine the bioactivity of native human TSP1 available from different commercial vendors (Thermo Scientific, Novus Biologicals, and Athens Research & Technology), specifically in terms of modulation of vasoactivity, in order to determine which TSP1 protein products can be used for the candidate drug screening assays afterwards.

Techniques: Isolation, Control, Concentration Assay, Produced, Inhibition

Journal: Methods and Protocols

Article Title: Efficient Ex Vivo Screening of Agents Targeting Thrombospondin1-Induced Vascular Dysfunction Using a Digital Multiwire Myograph System

doi: 10.3390/mps4040074

Figure Lengend Snippet: Thrombospondin 1 (TSP1)−impaired endothelium-dependent vasorelaxation was prevented by treatment with the proposed drug candidate, but not by blocking signal-regulatory protein α (SIRP α). Representative force vs. time traces recording from endothelium intactness test to acetylcholine-induced vasorelaxation by isolated murine thoracic aorta pre-treated with the proposed drug candidate 6.6 nM ( A ), CD47 antibody (CD47 ab) 1 µg/mL ( B ), or SIRPα antibody (SIRPα ab) 2 µg/mL ( C ) in the presence of Pierce TM —TSP1 4.4 nM (PE: phenylephrine; Ach: acetylcholine; the numbers marked in the recording are the logarithmic values of stimulant concentrations). Prior to receiving any treatment, the vessel segments were pre-contracted with 1 × 10 −6 M PE followed by 1 × 10 −6 M Ach-stimulated endothelium-dependent vasorelaxation to confirm endothelial intactness. ( D ) Concentration response curves of isolated mouse thoracic aorta exposed to TSP1, in the presence of vehicle, CD47-based drug candidate, or CD47 antibody. ( E ) Concentration response curves of isolated mouse thoracic aorta exposed to TSP1, in presence of vehicle or SIRPα antibody (***, p < 0.0001, TSP1, TSP1 + drug candidate, or TSP1 + SIRPα ab vs. control; ###, p < 0.0001, TSP1 + drug candidate or TSP1 + CD47 antibody vs. TSP1).

Article Snippet: Therefore, it was essential to examine the bioactivity of native human TSP1 available from different commercial vendors (Thermo Scientific, Novus Biologicals, and Athens Research & Technology), specifically in terms of modulation of vasoactivity, in order to determine which TSP1 protein products can be used for the candidate drug screening assays afterwards.

Techniques: Blocking Assay, Isolation, Concentration Assay, Control

Journal: Methods and Protocols

Article Title: Efficient Ex Vivo Screening of Agents Targeting Thrombospondin1-Induced Vascular Dysfunction Using a Digital Multiwire Myograph System

doi: 10.3390/mps4040074

Figure Lengend Snippet: Acute TSP1 challenge-induced reduction in endothelium-dependent vasorelaxation was significantly restored by therapeutic treatment with the proposed CD47-based drug candidate. Representative force vs. time traces recording from endothelium intactness test to acetylcholine-induced vasorelaxation by isolated murine thoracic aorta exposed to acute TSP1 (Pierce TM —TSP1 4.4 nM) challenge for 15 min, followed by treatment with the proposed CD47-based drug candidate 13.2 nM ( A ) or CD47 antibody (CD47 ab) 1 µg/mL ( B ) for 45 min (PE: phenylephrine; Ach: acetylcholine; the numbers marked in the recording are the logarithmic values of stimulant concentrations). Prior to receiving any treatment, the vessel segments were pre-contracted with 1 × 10 −6 M PE followed by 1 × 10 −6 M Ach-stimulated endothelium-dependent vasorelaxation to confirm endothelial intactness. ( C ) Concentration response curves of isolated mouse thoracic aorta exposed to TSP1, in the presence of vehicle, CD47-based drug candidate, or CD47 antibody (***, p < 0.0001, TSP1 or TSP1 + drug candidate vs. control; #, p < 0.05, TSP1 + drug candidate vs. TSP1; ###, p < 0.0001, TSP1 + CD47 antibody vs. TSP1).

Article Snippet: Therefore, it was essential to examine the bioactivity of native human TSP1 available from different commercial vendors (Thermo Scientific, Novus Biologicals, and Athens Research & Technology), specifically in terms of modulation of vasoactivity, in order to determine which TSP1 protein products can be used for the candidate drug screening assays afterwards.

Techniques: Isolation, Concentration Assay, Control

Journal: Frontiers in Immunology

Article Title: Integrated transcriptomic profiling of programmed cell death patterns unveils macrophage-hepatocyte crosstalk via THBS1-CD47 axis in hepatic ischemia-reperfusion injury

doi: 10.3389/fimmu.2026.1769849

Figure Lengend Snippet: OGD/R model induced THBS1 expression in RAW264.7 cells and apoptosis in AML12 cells. (A) Schematic of OGD/R treatment. AML12 cells (lower chamber) and RAW264.7 cells (upper chamber) were co-cultured in serum-free, glucose-free DMEM under hypoxic conditions (1% O2) for 6 hours, followed by re-oxygenation in complete medium for another 3 hours. Control cells were cultured in complete medium under normoxic conditions. (B) THBS1 protein expression in RAW264.7 cells between Control and OGD/R groups. (C) The contents of IL-1β, IL-6, and TNF-α in RAW264.7 cell culture medium between Control and OGD/R groups. (D) The expressions of pro-apoptotic proteins Bax, Cleaved-caspase 3, and anti-apoptotic Bcl2 protein in AML12 cells between Control and OGD/R groups. (E) Cell death in the Control and OGD/R groups was assessed using Hoechst 33342 and PI double staining. Scale bar = 50μm. (F) The mitochondrial membrane potential (ΔψM) of the Control and OGD/R groups was detected using JC-1 staining. Scale bar = 50μm.

Article Snippet: Furthermore, AML12 cells were seeded in 6-well plates 24 hours prior to the establishment of the OGD/R model, with each well receiving 1 μg/mL of recombinant mouse THBS1 protein (MCE, Shanghai, China).

Techniques: Expressing, Cell Culture, Control, Double Staining, Membrane, Staining

Journal: Frontiers in Immunology

Article Title: Integrated transcriptomic profiling of programmed cell death patterns unveils macrophage-hepatocyte crosstalk via THBS1-CD47 axis in hepatic ischemia-reperfusion injury

doi: 10.3389/fimmu.2026.1769849

Figure Lengend Snippet: RAW264.7-specific THBS1 siRNA treatment alleviated OGD/R-induced apoptosis in AML12 cells. (A) Schematic of OGD/R treatment. RAW264.7 cells, separately transfected with siRNA-NC and siRNA-THBS1, were seeded in the upper chamber and co-cultured with AML12 cells (lower chamber) under hypoxic conditions (1% O2) in serum-free, glucose-free DMEM for 6 hours, followed by reoxygenation in complete medium for another 3 hours. (B) THBS1 protein expression in RAW264.7 cells between siRNA-NC and siRNA-THBS1 groups. (C) The contents of IL-1β, IL-6, and TNF-α in RAW264.7 cell culture medium between siRNA-NC and siRNA-THBS1 groups. (D) The expression of pro-apoptotic proteins Bax, Cleaved-caspase 3, and anti-apoptotic Bcl2 protein in AML12 cells between siRNA-NC and siRNA-THBS1 groups. (E) Cell death in the siRNA-NC and siRNA-THBS1 groups was assessed using Hoechst 33342 and PI double staining. Scale bar = 50μm. (F) The mitochondrial membrane potential (ΔψM) of the siRNA-NC and siRNA-THBS1 groups was detected using JC-1 staining. Scale bar = 50μm.

Article Snippet: Furthermore, AML12 cells were seeded in 6-well plates 24 hours prior to the establishment of the OGD/R model, with each well receiving 1 μg/mL of recombinant mouse THBS1 protein (MCE, Shanghai, China).

Techniques: Transfection, Cell Culture, Expressing, Double Staining, Membrane, Staining

Journal: Frontiers in Immunology

Article Title: Integrated transcriptomic profiling of programmed cell death patterns unveils macrophage-hepatocyte crosstalk via THBS1-CD47 axis in hepatic ischemia-reperfusion injury

doi: 10.3389/fimmu.2026.1769849

Figure Lengend Snippet: THBS1 promoted hepatocyte apoptosis by inhibiting PI3K-AKT- NF-κB signaling. (A) The expression of p-AKT, AKT, p-NF-κB p65 and NF-κB p65 proteins in AML12 cells between siRNA-NC and siRNA-THBS1 groups. (B) The expression of pro-apoptotic proteins Bax, Cleaved-caspase 3, and anti-apoptotic Bcl2 protein in AML12 cells among the siRNA-NC, siRNA-THBS1 and siRNA-THBS1+ LY294002 groups. (C) Cell death in the siRNA-NC, siRNA-THBS1 and siRNA-THBS1+ LY294002 groups was assessed using Hoechst 33342 and PI double staining. Scale bar = 50μm. (D) The mitochondrial membrane potential (ΔψM) of the siRNA-NC, siRNA-THBS1 and siRNA-THBS1+ LY294002 was detected using JC-1 staining. Scale bar = 50μm.

Article Snippet: Furthermore, AML12 cells were seeded in 6-well plates 24 hours prior to the establishment of the OGD/R model, with each well receiving 1 μg/mL of recombinant mouse THBS1 protein (MCE, Shanghai, China).

Techniques: Expressing, Double Staining, Membrane, Staining

Journal: Frontiers in Immunology

Article Title: Integrated transcriptomic profiling of programmed cell death patterns unveils macrophage-hepatocyte crosstalk via THBS1-CD47 axis in hepatic ischemia-reperfusion injury

doi: 10.3389/fimmu.2026.1769849

Figure Lengend Snippet: THBS1-mediated hepatocyte apoptosis depended on the CD47 receptor. (A) CD47 protein expression in AML12 cells between Control and OGD/R groups. (B) Schematic of OGD/R treatment. AML12 cells, separately transfected with siRNA-NC and siRNA-CD47, were seeded in the lower chamber and co-cultured with RAW264.7 cells (upper chamber) under hypoxic conditions (1% O2) in serum-free, glucose-free DMEM for 6 hours, followed by reoxygenation in complete medium for another 3 hours. (C) The expression of pro-apoptotic proteins Bax, Cleaved-caspase 3, and anti-apoptotic Bcl2 protein in AML12 cells between siRNA-NC and siRNA-CD47 groups. (D) Cell death in the siRNA-NC and siRNA-CD47 groups was assessed using Hoechst 33342 and PI double staining. Scale bar = 50μm. (E) Schematic of OGD/R treatment. AML12 cells were separately transfected with siRNA-NC and siRNA-CD47, respectively, then cultured in serum-free, glucose-free DMEM supplemented with 1 ng/mL rmTHBS1 under hypoxic conditions (1% O2) for 6 hours, followed by reoxygenation in complete medium for another 3 hours. (F) The expression of pro-apoptotic proteins Bax, Cleaved-caspase 3, and anti-apoptotic Bcl2 protein in AML12 cells among the siRNA-NC, siRNA-NC+ rmTHBS1 and siRNA-CD47+ rmTHBS1 groups. (G) Cell death in the siRNA-NC, siRNA-NC+ rmTHBS1 and siRNA-CD47+ rmTHBS1 groups was assessed using Hoechst 33342 and PI double staining. Scale bar = 50μm. (H) Schematic diagram of the THBS1-CD47 axis’s role in macrophage-hepatocyte crosstalk during HIRI.

Article Snippet: Furthermore, AML12 cells were seeded in 6-well plates 24 hours prior to the establishment of the OGD/R model, with each well receiving 1 μg/mL of recombinant mouse THBS1 protein (MCE, Shanghai, China).

Techniques: Expressing, Control, Transfection, Cell Culture, Double Staining

Journal: Investigative Ophthalmology & Visual Science

Article Title: Corneal Nerves Promote Alkali Burn Repair by Modulating Macrophages and Neutrophils via Calcitonin Gene-Related Peptide

doi: 10.1167/iovs.67.4.60

Figure Lengend Snippet: CGRP modulates macrophage function via the cAMP/TSP-1 signaling pathway ( n = 4). (A) Schematic of the cAMP signaling pathway downstream of the CGRP-CALCRL/RAMP1 axis. Activation of adenylyl cyclase (AC) elevates the intracellular level of cAMP. (B) The concentration of cAMP in cell lysates was measured by a competitive ELISA after in vitro stimulation of macrophages with CGRP or co-stimulation of CGRP with SQ22536 for 30 minutes ( n = 4). (C) Bar graph showing quantification of apoptosis in macrophages following in vitro treatment with CGRP alone or in combination with SQ22536 ( n = 4). (D, E) The expression of CD206 and Arg-1 in macrophages after treatment with CGRP or CGRP combined with SQ22536 ( n = 4). (F) Macrophage efferocytosis after CGRP or CGRP + SQ22536 treatment ( n = 4). (G) Volcano plot showing upregulation of thbs1 gene expression in CGRP-treated macrophages. (H, J) Representative micrographs and quantification of increased TSP-1 expression in macrophages following CGRP or CGRP + SQ22536 stimulation in vitro ( n = 4). (I, K) Representative micrographs and quantification of TSP-1+ macrophages in the cornea following 7 days of topical CGRP treatment ( n = 4). (L) Quantification of apoptosis in macrophages treated with TSP-1 in vitro ( n = 4). (M, N) The expression of CD206 and Arg-1 in macrophages after treatment with TSP-1 ( n = 4). (O) Macrophage efferocytosis after CGRP or CGRP + SQ22536 treatment. Data are shown as mean ± SD. ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: For TSP-1-induced apoptosis, BMDMs were incubated with RPMI-1640 medium containing 100 nM TSP-1 (HY-P701325; MedChenExpress) for 24 hours.

Techniques: Activation Assay, Concentration Assay, Competitive ELISA, In Vitro, Expressing, Gene Expression

Journal: bioRxiv

Article Title: The secreted tyrosine kinase VLK is essential for normal platelet activation and thrombus formation

doi: 10.1101/2021.01.24.427281

Figure Lengend Snippet: (A) Immunoblots of lysates (Lys.) and supernatants (Sup.) from Vlk f/f (CTL) and Vlk-cKO (cKO) platelets resting or stimulated with 5 U/mL thrombin for 15 minutes. Anti-Tsp1 and anti-PF4 antibodies were used as markers of platelet activation, and anti-GAPDH antibody was used to examine total protein levels in lysates. (B) Lysates from A were used to examine tyrosine phosphoproteins by immunoblot (IB) of p-Tyr enriched fraction (IP). Black arrows indicate phopshobands decreased in Vlk-cKO (cKO) compared to Vlk f/f (CTL) in resting (a-f) and activated (1-10) platelets. (C, D) Cumulative percent tyrosine phosphorylation in phosphobands indicated in representative figure in B from resting ( C ) and activated ( D ) control and Vlk-cKO lysates; data are expressed as mean ± SD; n=3 independent experiments. (E) Representative image of input and p-Tyr IP fraction from resting and thrombin-stimulated supernatants analyzed by SDS-PAGE with 8% gels and immunoblotting with anti-p-Tyr and anti-Tsp1 antibodies. Phenylphosphate (PP) was used as negative control for non-specific binding to p-Tyr beads. Black arrows indicate bands decreasing in cKO compared to CTL. (F) Cumulative percent phosphorylation of Tsp1 in activated supernatants from Vlk-cKO compared to CTL; n=3 independent experiments.

Article Snippet: The following primary antibodies were used for protein immunoblots: Anti p-Tyr (4G10, Millipore Sigma, #05-321 and P-Tyr-1000, Cell Signaling Technology, #8954), anti V5-HRP (ThermoFisher Scientific, #46-0708), anti ENTPD6 (GeneTex, #GTX101851), anti VLK raised against epitope SRAEYQRIPDSAITQEDYR of mouse VLK (Abfrontier), anti p-VLK (Y64) raised against epitope GRGELARQIRERYEEVQRYSRG phosphorylated at Y64 of mouse VLK (Abfrontier), anti cytoplasmic actin (Milipore Sigma, #A4700), anti GAPDH-HRP (GeneTex, #GTX627408-01), anti p-Akt (S473) (Cell Signaling Technology, #4060), anti Akt (Cell Signaling Technology, #4691), anti p-Erk 1/2 (T202/Y204) (Cell Signaling Technology, #4370), anti Erk 1/2 (Cell Signaling Technology, #4695), anti P-selectin (R&D Systems, #AF737), anti ITGβ3 (Cell Signaling Technology, #13166), anti ITGα2b (Boster, #PB9647), anti FGNγ (GeneTex, #GTX108640), anti vWR (Dako, #A0082 and Santa Cruz Biotechnology, #sc-271409), anti Platelet Factor 4 (PF4) (R&D Systems, #AF595), anti GPIbɑ (Emfret Analytics, #M043-0), and anti thrombospondin 1 (Tsp1 ) (Bethyl, #A304-989A-M).

Techniques: Western Blot, Activation Assay, Phospho-proteomics, Control, SDS Page, Negative Control, Binding Assay

Journal: bioRxiv

Article Title: The secreted tyrosine kinase VLK is essential for normal platelet activation and thrombus formation

doi: 10.1101/2021.01.24.427281

Figure Lengend Snippet: (A) Evaluation of aggregation of washed platelets (2×10 8 platelets/ml) from control mice ( grey ) and platelet-specific VLK knockout mice ( blue ) stimulated with 100 μM PAR4 peptide AYPGKF. Representative tracings are shown in left panel and cumulative data (n = 3-5) in right panel . ( B ) Platelets from control mice ( grey ) and VLK knockout mice ( blue ) were stimulated with 4 μg/mL collagen and evaluated as in A . (C) Expression of P-selectin in platelets from control ( grey ) and platelet-specific VLK knockout mice ( blue ) in response to AYPGKF. (D) Protein immunoblots of lysates (Lys.) and supernatants (Sup.) from Vlk f/f (CTL) and Vlk-cKO (cKO) platelets resting or stimulated with 100 μM PAR4 peptide AYPGKF for 15 minutes. Anti-Tsp1 and anti-PF4 antibodies were used to detect secretion of α-granule cargo, and anti-GAPDH antibody was used to determine total protein levels in lysates. ( E ) Concurrent monitoring of aggregation ( blue trac ing) and ATP release ( red tracing ) following exposure to 50 μM AYPGKF in platelets from control ( left panel ) and platelet-specific VLK knockout mice ( right panel ). (F) Aggregation studies in platelets from control ( left panel ) and platelet-specific VLK knockout mice ( right panel ) were performed as described in E except that ADP 2.5 μM was added together with AYPGKF.

Article Snippet: The following primary antibodies were used for protein immunoblots: Anti p-Tyr (4G10, Millipore Sigma, #05-321 and P-Tyr-1000, Cell Signaling Technology, #8954), anti V5-HRP (ThermoFisher Scientific, #46-0708), anti ENTPD6 (GeneTex, #GTX101851), anti VLK raised against epitope SRAEYQRIPDSAITQEDYR of mouse VLK (Abfrontier), anti p-VLK (Y64) raised against epitope GRGELARQIRERYEEVQRYSRG phosphorylated at Y64 of mouse VLK (Abfrontier), anti cytoplasmic actin (Milipore Sigma, #A4700), anti GAPDH-HRP (GeneTex, #GTX627408-01), anti p-Akt (S473) (Cell Signaling Technology, #4060), anti Akt (Cell Signaling Technology, #4691), anti p-Erk 1/2 (T202/Y204) (Cell Signaling Technology, #4370), anti Erk 1/2 (Cell Signaling Technology, #4695), anti P-selectin (R&D Systems, #AF737), anti ITGβ3 (Cell Signaling Technology, #13166), anti ITGα2b (Boster, #PB9647), anti FGNγ (GeneTex, #GTX108640), anti vWR (Dako, #A0082 and Santa Cruz Biotechnology, #sc-271409), anti Platelet Factor 4 (PF4) (R&D Systems, #AF595), anti GPIbɑ (Emfret Analytics, #M043-0), and anti thrombospondin 1 (Tsp1 ) (Bethyl, #A304-989A-M).

Techniques: Control, Knock-Out, Expressing, Western Blot

Journal: bioRxiv

Article Title: The secreted tyrosine kinase VLK is essential for normal platelet activation and thrombus formation

doi: 10.1101/2021.01.24.427281

Figure Lengend Snippet: (A) Representative image of p-Akt (S473) and total Akt levels in lysates from CTL and cKO platelets resting or stimulated with 100 μM AYPGKF. (B) Cumulative percent phosphorylation of S473 in Akt; n=3 independent experiments. (C) Representative image of p-Erk 1/2 (T202/Y204) and Erk 1/2 total levels in lysates from CTL and cKO platelets resting or stimulated with 100 μM AYPGKF. (D) Cumulative percent phosphorylation of Erk 1/2 ; n=3 independent experiments. ( E ) Proposed model for the role of VLK in platelet function. Step 1, stimulation through PAR4 by a submaximal agonist concentration results in partial activation of kinases involved in dense granule release, such as Akt and Erk 1/2 . Step 2, signaling downstream of Akt and Erk 1/2 results in release of ADP from dense granules. VLK appears to act in these steps leading to dense granule release by mechanisms that are yet to be determined but could include phosphorylation of receptors on extracellular domains, phosphorylation of secretory chaperones, or phosphorylation of dense granule resident proteins important for release. Step 3, ADP released from dense granules stimulates purinergic receptors, enhancing signaling through kinases involved in granule release. Step 4, exocytosis of α-granules results in the release of VLK into the extracellular environment along with ATP and VLK substrates, such as thrombospondin 1.

Article Snippet: The following primary antibodies were used for protein immunoblots: Anti p-Tyr (4G10, Millipore Sigma, #05-321 and P-Tyr-1000, Cell Signaling Technology, #8954), anti V5-HRP (ThermoFisher Scientific, #46-0708), anti ENTPD6 (GeneTex, #GTX101851), anti VLK raised against epitope SRAEYQRIPDSAITQEDYR of mouse VLK (Abfrontier), anti p-VLK (Y64) raised against epitope GRGELARQIRERYEEVQRYSRG phosphorylated at Y64 of mouse VLK (Abfrontier), anti cytoplasmic actin (Milipore Sigma, #A4700), anti GAPDH-HRP (GeneTex, #GTX627408-01), anti p-Akt (S473) (Cell Signaling Technology, #4060), anti Akt (Cell Signaling Technology, #4691), anti p-Erk 1/2 (T202/Y204) (Cell Signaling Technology, #4370), anti Erk 1/2 (Cell Signaling Technology, #4695), anti P-selectin (R&D Systems, #AF737), anti ITGβ3 (Cell Signaling Technology, #13166), anti ITGα2b (Boster, #PB9647), anti FGNγ (GeneTex, #GTX108640), anti vWR (Dako, #A0082 and Santa Cruz Biotechnology, #sc-271409), anti Platelet Factor 4 (PF4) (R&D Systems, #AF595), anti GPIbɑ (Emfret Analytics, #M043-0), and anti thrombospondin 1 (Tsp1 ) (Bethyl, #A304-989A-M).

Techniques: Phospho-proteomics, Concentration Assay, Activation Assay