Journal: Frontiers in Cell and Developmental Biology

Article Title: Thrombospondin-4 Is a Soluble Dermal Inflammatory Signal That Selectively Promotes Fibroblast Migration and Keratinocyte Proliferation for Skin Regeneration and Wound Healing

doi: 10.3389/fcell.2021.745637

Figure Lengend Snippet: THBS4 expression in human skin following burn injury. (A) Normal skin from healthy controls; (B) skin from burn injury patients. Biopsy samples were collected from the study area at 3, 14, and 21 days after the wound excision. E—epidermis. 3 representative samples in each group are shown. Scale bar is 200 μm. (C) Relative quantification of THBS4 expression by mean integrated density of the fluorescence signal. The plot depicts the distribution of 10 samples, * indicates a statistically significant ( P < 0.05) difference.

Article Snippet: To generate THBS4 -expression plasmid, human THBS4 sequence was amplified from ORF cDNA clone expression plasmid (catalog no HGI8843-UT, Sino Biological, Beijing, China).

Techniques: Expressing, Fluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: Thrombospondin-4 Is a Soluble Dermal Inflammatory Signal That Selectively Promotes Fibroblast Migration and Keratinocyte Proliferation for Skin Regeneration and Wound Healing

doi: 10.3389/fcell.2021.745637

Figure Lengend Snippet: THBS4 is upregulated in regenerating mouse skin. Full-thickness dermal wounds were generated in mouse dorsal skin. (A) THBS4 expression was characterized by immunofluorescence microscopy in healthy skin and at 2-, 4-, 6-, and 8-days post wounding. 3 representative samples in each group are shown. Yellow arrows indicate THBS4 expression in hair follicles. Scale bar is 200 μm. (B) Relative quantification of THBS4 expression by mean integrated density of the fluorescence signal. Bars show the average of 3 samples for each time point ± standard deviation, * indicates a statistically significant ( P < 0.05) difference.

Article Snippet: To generate THBS4 -expression plasmid, human THBS4 sequence was amplified from ORF cDNA clone expression plasmid (catalog no HGI8843-UT, Sino Biological, Beijing, China).

Techniques: Generated, Expressing, Immunofluorescence, Microscopy, Fluorescence, Standard Deviation

Journal: Frontiers in Cell and Developmental Biology

Article Title: Thrombospondin-4 Is a Soluble Dermal Inflammatory Signal That Selectively Promotes Fibroblast Migration and Keratinocyte Proliferation for Skin Regeneration and Wound Healing

doi: 10.3389/fcell.2021.745637

Figure Lengend Snippet: THBS4 expression in healthy control skin and psoriatic skin lesions. E—epidermis; D—dermis. 3 representative samples in each group are shown (A) and relative quantification of THBS4 expression by mean integrated density of the fluorescence signal (B) , n = 5. Scale bar is 200 μm. (C) THBS4 co-localization with fibroblast marker vimentin (Vim) and integrin beta 4 (ITGB4). Scale bar is 50 μm. The plot depicts the distribution of 5 samples, * indicates a statistically significant ( P < 0.05) difference.

Article Snippet: To generate THBS4 -expression plasmid, human THBS4 sequence was amplified from ORF cDNA clone expression plasmid (catalog no HGI8843-UT, Sino Biological, Beijing, China).

Techniques: Expressing, Fluorescence, Marker

Journal: Frontiers in Cell and Developmental Biology

Article Title: Thrombospondin-4 Is a Soluble Dermal Inflammatory Signal That Selectively Promotes Fibroblast Migration and Keratinocyte Proliferation for Skin Regeneration and Wound Healing

doi: 10.3389/fcell.2021.745637

Figure Lengend Snippet: THBS4 promotes fibroblast migration. (A) Transwell migration assay with fibroblasts stimulated with recombinant THBS4 and quantification of the number of migrating cells in a field of view. (B) Transwell invasion assay through Matrigel-coated chambers and quantification of the number of migrating cells in a field of view. (C) Representative images of the in vitro scratch wound healing assay with fibroblasts stimulated with recombinant THBS4 and quantification of the relative wound closure in time. Scale bar is 200 μm. The graphs depict the averages of at least 3 independent replicates ± standard deviation, * indicates a statistically significant ( P < 0.05) difference compared to cells stimulated with control medium.

Article Snippet: To generate THBS4 -expression plasmid, human THBS4 sequence was amplified from ORF cDNA clone expression plasmid (catalog no HGI8843-UT, Sino Biological, Beijing, China).

Techniques: Migration, Transwell Migration Assay, Recombinant, Transwell Invasion Assay, In Vitro, Wound Healing Assay, Standard Deviation

Journal: Frontiers in Cell and Developmental Biology

Article Title: Thrombospondin-4 Is a Soluble Dermal Inflammatory Signal That Selectively Promotes Fibroblast Migration and Keratinocyte Proliferation for Skin Regeneration and Wound Healing

doi: 10.3389/fcell.2021.745637

Figure Lengend Snippet: THBS4 promotes keratinocyte but not fibroblast proliferation in vitro . (A) Representative images of primary human fibroblasts cultured in the presence of recombinant THBS4 protein and the quantification of Ki67 + positive cells, n = 3 (B) . (C) Representative images of primary human keratinocytes cultured in the presence of recombinant THBS4 protein and quantification of Ki67 + positive cells. (D) Scale bar is 200 μm. The graphs depict the averages of at least 3 independent replicates ± standard deviation, * indicates a statistically significant ( P < 0.05) difference compared to cells stimulated with control medium.

Article Snippet: To generate THBS4 -expression plasmid, human THBS4 sequence was amplified from ORF cDNA clone expression plasmid (catalog no HGI8843-UT, Sino Biological, Beijing, China).

Techniques: In Vitro, Cell Culture, Recombinant, Standard Deviation

Journal: Frontiers in Cell and Developmental Biology

Article Title: Thrombospondin-4 Is a Soluble Dermal Inflammatory Signal That Selectively Promotes Fibroblast Migration and Keratinocyte Proliferation for Skin Regeneration and Wound Healing

doi: 10.3389/fcell.2021.745637

Figure Lengend Snippet: Heatmap of differentially enriched genes in fibroblasts in response to THBS4 stimulation. Statistically significant ( P < 0.05) and expression fold-change > 2 protein coding genes and microRNAs are shown, n = 2.

Article Snippet: To generate THBS4 -expression plasmid, human THBS4 sequence was amplified from ORF cDNA clone expression plasmid (catalog no HGI8843-UT, Sino Biological, Beijing, China).

Techniques: Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Thrombospondin-4 Is a Soluble Dermal Inflammatory Signal That Selectively Promotes Fibroblast Migration and Keratinocyte Proliferation for Skin Regeneration and Wound Healing

doi: 10.3389/fcell.2021.745637

Figure Lengend Snippet: Ingenuity Pathway Analysis of the changes in fibroblast transcription profile in response to THBS4 stimulation. Causal network showing Frizzled and β-catenin 1 pathway activation is shown. Upper values indicate log2 fold changes, lower values show the P -value for the RNAseq.

Article Snippet: To generate THBS4 -expression plasmid, human THBS4 sequence was amplified from ORF cDNA clone expression plasmid (catalog no HGI8843-UT, Sino Biological, Beijing, China).

Techniques: Activation Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Thrombospondin-4 Is a Soluble Dermal Inflammatory Signal That Selectively Promotes Fibroblast Migration and Keratinocyte Proliferation for Skin Regeneration and Wound Healing

doi: 10.3389/fcell.2021.745637

Figure Lengend Snippet: Proteotranscriptomic analysis of THBS4 stimulation in fibroblasts. The combined network of transcriptomics analysis at 4 h stimulation and proteomics analysis at 24 h is shown. Single numbers indicate log2FC in proteomics and duplicate values indicate log2FC and P -value for RNAseq.

Article Snippet: To generate THBS4 -expression plasmid, human THBS4 sequence was amplified from ORF cDNA clone expression plasmid (catalog no HGI8843-UT, Sino Biological, Beijing, China).

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: Thrombospondin-4 Is a Soluble Dermal Inflammatory Signal That Selectively Promotes Fibroblast Migration and Keratinocyte Proliferation for Skin Regeneration and Wound Healing

doi: 10.3389/fcell.2021.745637

Figure Lengend Snippet: THBS4 promotes wound healing in vivo . Full dermal wounds were inflicted on the dorsal skin of C57/Bl6 mice and either recombinant THBS4 or PBS was applied. Representative images of the wounds from indicated time points (A) and measurements of the wound area (B) . The graph depicts the averages of 10 biological replicates ± standard deviation, * indicates a statistically significant ( P < 0.05) difference.

Article Snippet: To generate THBS4 -expression plasmid, human THBS4 sequence was amplified from ORF cDNA clone expression plasmid (catalog no HGI8843-UT, Sino Biological, Beijing, China).

Techniques: In Vivo, Recombinant, Standard Deviation

Journal: Frontiers in Cell and Developmental Biology

Article Title: Thrombospondin-4 Is a Soluble Dermal Inflammatory Signal That Selectively Promotes Fibroblast Migration and Keratinocyte Proliferation for Skin Regeneration and Wound Healing

doi: 10.3389/fcell.2021.745637

Figure Lengend Snippet: THBS4 is a soluble dermal inflammatory signal that activates the fibroblast migration for skin regeneration and wound healing. See section “Discussion” for closer explanation.

Article Snippet: To generate THBS4 -expression plasmid, human THBS4 sequence was amplified from ORF cDNA clone expression plasmid (catalog no HGI8843-UT, Sino Biological, Beijing, China).

Techniques: Migration

Journal: Respiratory Research

Article Title: The role and mechanism of thrombospondin-4 in pulmonary arterial hypertension associated with congenital heart disease

doi: 10.1186/s12931-024-02932-w

Figure Lengend Snippet: Circulating level of THBS4 in patients with PAH-CHD. The resultant data are represented as mean ± SD. * P < 0.05

Article Snippet: A human THBS4 ELISA kit (Cusabio, China) was used to measure the circulating level of THBS4 according to the manufacturer’s instructions.

Techniques:

Journal: Respiratory Research

Article Title: The role and mechanism of thrombospondin-4 in pulmonary arterial hypertension associated with congenital heart disease

doi: 10.1186/s12931-024-02932-w

Figure Lengend Snippet: Expression of THBS4 in PAH rats induced by MCT plus aorto-caval shunt. (A) THBS4 expression in GSE149899. qRT-PCR (B) and Western blot (C) showed the time course of THBS4 expression in PAH rats induced by MCT plus aorto-caval shunt. (D) Double immunofluorescence staining of THBS4 with α-SMA or VWF. Scare bars: 50 μm. CON: the control group; SHAM: the sham group; MS: PAH rats induced by MCT plus aorto-caval shunt (MS) from 1 week (1w) to 4 weeks (4w). The resultant data are represented as mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001

Article Snippet: A human THBS4 ELISA kit (Cusabio, China) was used to measure the circulating level of THBS4 according to the manufacturer’s instructions.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Double Immunofluorescence Staining, Control

Journal: Respiratory Research

Article Title: The role and mechanism of thrombospondin-4 in pulmonary arterial hypertension associated with congenital heart disease

doi: 10.1186/s12931-024-02932-w

Figure Lengend Snippet: qRT-PCR (A) and Western blot (B) showed the transfection efficiency of THBS4 in PASMCs. (C) The influence of THBS4 on the transformation of phenotype of PASMCs. The resultant data are represented as mean ± SD. *: si-THBS4 vs.si-CON; #: pEX3-THBS4 vs. pEX3-CON. * P < 0.05; *** P < 0.001; # P < 0.05; ## P < 0.01; ### P < 0.001

Article Snippet: A human THBS4 ELISA kit (Cusabio, China) was used to measure the circulating level of THBS4 according to the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Western Blot, Transfection, Transformation Assay

Journal: Respiratory Research

Article Title: The role and mechanism of thrombospondin-4 in pulmonary arterial hypertension associated with congenital heart disease

doi: 10.1186/s12931-024-02932-w

Figure Lengend Snippet: Effect of THBS4 on the proliferation, apoptosis, and migration of PASMCs. (A) Effect of THBS4 on proliferation of PASMCs, as determined by the EdU assay. Scare bars, 100 μm. (B) Effect of THBS4 on apoptosis of PASMCs, as determined by the Annexin-V flow cytometry. (C) Effect of THBS4 on migration of PASMCs, as determined by the Wound healing assay. Scare bars, 200 μm. (D) Effect of THBS4 on migration of PASMCs, as determined by the Transwell assay. Scare bars, 100 μm. The resultant data are represented as mean ± SD. *: si-THBS4 vs.si-CON; #: pEX3-THBS4 vs. pEX3-CON. * P < 0.05; ** P < 0.01; **** P < 0.0001; # P < 0.05; ## P < 0.01

Article Snippet: A human THBS4 ELISA kit (Cusabio, China) was used to measure the circulating level of THBS4 according to the manufacturer’s instructions.

Techniques: Migration, EdU Assay, Flow Cytometry, Wound Healing Assay, Transwell Assay

Journal: Respiratory Research

Article Title: The role and mechanism of thrombospondin-4 in pulmonary arterial hypertension associated with congenital heart disease

doi: 10.1186/s12931-024-02932-w

Figure Lengend Snippet: Phosphorylation level of PI3K/AKT in PASMCs after inhibition and overexpression of THBS4 in PASMCs. The resultant data are represented as mean ± SD. *: si-THBS4 vs. si-CON; #: pEX3-THBS4 vs. pEX3-CON. * P < 0.5; # P < 0.5

Article Snippet: A human THBS4 ELISA kit (Cusabio, China) was used to measure the circulating level of THBS4 according to the manufacturer’s instructions.

Techniques: Phospho-proteomics, Inhibition, Over Expression

Journal: Respiratory Research

Article Title: The role and mechanism of thrombospondin-4 in pulmonary arterial hypertension associated with congenital heart disease

doi: 10.1186/s12931-024-02932-w

Figure Lengend Snippet: Effect of THBS4 suppression on pulmonary vascular remodeling in PAH rats induced by MCT plus aorto-caval shunt. (A) The AAV transfection efficiency was determined by detecting green fluorescence protein which is encoded by AAV in lung tissues. Scare bars, 100 μm. (B) THBS4 suppression efficiency determined by immunofluorescence staining of THBS4. Scare bars, 50 μm. (C) Hematoxylin and Eosin staining of lung tissues. Scare bars,100 μm. (D) Immunofluorescence staining of α-SMA in lung tissues. Scare bars,100 μm. The percentage of non-muscularized (E) , partially-muscularized (F), and fully-muscularized pulmonary arterioles (G) . (H) The percentage of wall thickness. (I) The percentage of wall area. (G) Cross-sectional area of the right ventricular cardiomyocyte. Scare bars,100 μm. The resultant data are represented as mean ± SD. CON: control group; SHAM: sham group; MS-AAV-CON: administration of control AAV + MCT plus aorto-caval shunt induced PAH rats; MS-AAV-siTHBS4: administration of THBS4 suppression AAV + MCT plus aorto-caval shunt induced PAH rats. PA: pulmonary arteriole. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001

Article Snippet: A human THBS4 ELISA kit (Cusabio, China) was used to measure the circulating level of THBS4 according to the manufacturer’s instructions.

Techniques: Transfection, Fluorescence, Immunofluorescence, Staining, Control

Journal: Cell Death & Disease

Article Title: Effects of thrombospondin-4 on pro-inflammatory phenotype differentiation and apoptosis in macrophages

doi: 10.1038/s41419-020-2237-2

Figure Lengend Snippet: Cultured RAW264.7 were treated with pro-inflammatory (1000 IU/ml INF-γ, 20 ng/ml GM-CSF, 0.5 μg/ml LPS) and anti-inflammatory (40 ng/ml IL-4, 20 ng/ml M-CSF) stimuli for indicated times. a TSP-4 protein was detected in western blotting with anti-TSP-4 Ab, representative images. b Protein levels were quantified by densitometry; n = 3; p < 0.05. c Thbs4 expression was measured by QRT-PCR; n = 3; * p < 0.05. Cultured BMDM were treated with pro-inflammatory (1000 IU/ml INF-γ, 20 ng/ml GM-CSF, 0.5 μg/ml LPS) and anti-inflammatory (40 ng/ml IL-4, 20 ng/ml M-CSF) stimuli for indicated times. d TSP-4 protein was detected in western blotting with anti-TSP-4 Ab, representative images. e Protein levels were quantified by densitometry; n = 3; * p < 0.05. f Thbs4 expression was measured by QRT-PCR; n = 3; * p < 0.05.

Article Snippet: The TaqMan ® probe for Thbs4 (Assay ID# Mm03003598_s1, ThermoFisher), CD68 (Assay ID# Mm03047343_m1, ThermoFisher), CD38 (Assay ID# Mm01220906_m1, ThermoFisher), Egr2 (Assay ID# Mm00456650_m1, ThermoFisher), Nos2 (Assay ID# Mm00440502_m1, ThermoFisher), Arg1 (Assay ID# Mm00475988_m1, ThermoFisher) were used for detecting respective genes expressions with Stx5a (Assay ID# Mm00502335_m1, ThermoFisher)) and ACTB (Assay ID# Mm02619580_g1, ThermoFisher) were used as housekeeping gene controls.

Techniques: Cell Culture, Western Blot, Expressing, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: Effects of thrombospondin-4 on pro-inflammatory phenotype differentiation and apoptosis in macrophages

doi: 10.1038/s41419-020-2237-2

Figure Lengend Snippet: a BMDM were treated with LPS (0.5 μg/mL) and IFNγ (1000 IU/mL) for 6 h, and Nos2 and CD38 mRNA expression was measured by QRT-PCR; fold decrease is shown on the y -axis (control = 1 in each genotype) n = 3; * p < 0.05. b In all, 5 μg/ml of recombinant TSP-4 (rTSP-4) was used to stimulate BMDM for 6 h, and Nos2 and CD38 mRNA expression was measured by QRT-PCR; n = 3; * p < 0.05. c Cultured BMDM from WT and Thbs4 − / − mice were stimulated for 24 h with IL-4 or M-CSF: * p < 0.05 compared with WT Control (no stimulation); # p < 0.05 compared with KO Control (no stimulation), $ p < 0.05 compared with identical treatment in WT cells.

Article Snippet: The TaqMan ® probe for Thbs4 (Assay ID# Mm03003598_s1, ThermoFisher), CD68 (Assay ID# Mm03047343_m1, ThermoFisher), CD38 (Assay ID# Mm01220906_m1, ThermoFisher), Egr2 (Assay ID# Mm00456650_m1, ThermoFisher), Nos2 (Assay ID# Mm00440502_m1, ThermoFisher), Arg1 (Assay ID# Mm00475988_m1, ThermoFisher) were used for detecting respective genes expressions with Stx5a (Assay ID# Mm00502335_m1, ThermoFisher)) and ACTB (Assay ID# Mm02619580_g1, ThermoFisher) were used as housekeeping gene controls.

Techniques: Expressing, Quantitative RT-PCR, Control, Recombinant, Cell Culture

Journal: Cell Death & Disease

Article Title: Effects of thrombospondin-4 on pro-inflammatory phenotype differentiation and apoptosis in macrophages

doi: 10.1038/s41419-020-2237-2

Figure Lengend Snippet: a Bone-marrow-derived macrophages (BMDM) from WT, KO, and KI mice were differentiated in culture in M1 or M2 differentiation media, and the number of survived cells (number of cells on day 5/number of cells 3 h after seeding) was measured using the CyQuant cell survival assay kit; n = 5; * p <0.05. b Purified mouse blood monocytes were differentiated in M1 or M2 differentiation cell culture media, and the number of survived cells was measured; n = 12; * p <0.05. c BMDM were differentiated in M1 differentiation medium for 7 days, and stimulated with LPS for 48 h. The expression of Bax , Bcl2 , and Casp3 and the activity of Caspase 3/7 enzymes were measured in cultured BMDM from WT and Thbs4 −/− mice; n = 3; * p < 0.05. d The activity of Caspase 3/7 enzymes was measured in cultured BMDM from WT and P387-TSP-4-KI mice; n = 3; * p < 0.05. e basal levels of expression of WT A387-TSP-4 and mutant P387-TSP-4 in cultured BMDM isolated from WT and P387-TSP-4-KI mice. Western blotting with anti-TSP-4 and anti-β-actin antibodies.

Article Snippet: The TaqMan ® probe for Thbs4 (Assay ID# Mm03003598_s1, ThermoFisher), CD68 (Assay ID# Mm03047343_m1, ThermoFisher), CD38 (Assay ID# Mm01220906_m1, ThermoFisher), Egr2 (Assay ID# Mm00456650_m1, ThermoFisher), Nos2 (Assay ID# Mm00440502_m1, ThermoFisher), Arg1 (Assay ID# Mm00475988_m1, ThermoFisher) were used for detecting respective genes expressions with Stx5a (Assay ID# Mm00502335_m1, ThermoFisher)) and ACTB (Assay ID# Mm02619580_g1, ThermoFisher) were used as housekeeping gene controls.

Techniques: Derivative Assay, CyQUANT Assay, Clonogenic Cell Survival Assay, Purification, Cell Culture, Expressing, Activity Assay, Mutagenesis, Isolation, Western Blot

Journal: Cell Death & Disease

Article Title: Effects of thrombospondin-4 on pro-inflammatory phenotype differentiation and apoptosis in macrophages

doi: 10.1038/s41419-020-2237-2

Figure Lengend Snippet: a Expression of CD68, CD38, and Egr-2 was measured in peritoneal tissues of WT mice with LPS-induced peritonitis; n = 3; * p <0.05. b Expression of CD68 and CD38 was measured in peritoneal tissues of Thbs4 − / − mice with LPS-induced peritonitis; n = 3; * p < 0.05. c Expression of CD68 and CD38 was measured in peritoneal tissues of P387-TSP-4-KI mice with LPS-induced peritonitis; n = 3; * p < 0.05. d Migration of RAW264.7 was measured in Boyden chambers (uncoated, coated with A387 or P387 rTSP-4, or BSA); n = 3; * p < 0.05 compared with plastic and BSA; # p < 0.05 compared with WT rTSP-4. e A387 and P387 rTSP-4 were used in adhesion competition assay: plastic was coated with P387 rTSP-4, and RAW264.7 was added in increasing concentrations; n = 3; * p < 0.05.

Article Snippet: The TaqMan ® probe for Thbs4 (Assay ID# Mm03003598_s1, ThermoFisher), CD68 (Assay ID# Mm03047343_m1, ThermoFisher), CD38 (Assay ID# Mm01220906_m1, ThermoFisher), Egr2 (Assay ID# Mm00456650_m1, ThermoFisher), Nos2 (Assay ID# Mm00440502_m1, ThermoFisher), Arg1 (Assay ID# Mm00475988_m1, ThermoFisher) were used for detecting respective genes expressions with Stx5a (Assay ID# Mm00502335_m1, ThermoFisher)) and ACTB (Assay ID# Mm02619580_g1, ThermoFisher) were used as housekeeping gene controls.

Techniques: Expressing, Migration, Competitive Binding Assay