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R&D Systems anti timp1 antibody af980

Fig. 1. TIMP-1fl/fl knockout mice develop a severe EAE phenotype that is immunologically independent. (A) Schematic of design of development of the flox-targeted sites that enabled cell and tissue-specific deletion of <t>TIMP-1</t> in GFAP-Cre-TIMP-1fl/fl mice. (B) Immunocytochemical validation of CRE expression in astrocytes from GFAP-Cre:TIMP-1fl/fl mice, and (C) physiological validation of TIMP-1 mRNA expression in primary astrocytes and absence of TIMP-1 mRNA expression in astrocyte cultures from GFAP-Cre:TIMP-1fl/fl mice (n = 8 per treatment; ANOVA; ****P < 0.0001; where *P < 0.01; ***P < 0.002). (D) Validation of absent TIMP-1 protein expression in primary astrocyte cultures from GFAP-Cre:Timp1fl/f mice (n = 3 biological replicates). (E) Clinical EAE in MOG35-55-treated C57Bl/6 WT and GFAP-Cre:TIMP-1fl/fl mice over 52 d time course (n = 13 to 17/treatment and genotype; 2-way ANOVA, P < 0.0001, where post-hoc multiple-comparisons identified significance at *P < 0.05 and **P < 0.01, as indicated). (F and G) Flow cytometry analysis of overall CD4+/IFNγ+ T cells from spinal cords of Wt and GFAP-Cre:TIMP- 1fl/fl mice at peak clinical EAE illness (day 18; n = 3 per genotype and treatment), including (H) analysis of CD4+/IFNγ+ T cells (t test, unpaired t test, P < 0.20).

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1) Product Images from "Astrocytic TIMP-1 regulates production of Anastellin, an inhibitor of oligodendrocyte differentiation and FTY720 responses."

Article Title: Astrocytic TIMP-1 regulates production of Anastellin, an inhibitor of oligodendrocyte differentiation and FTY720 responses.

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.2306816121

Figure Legend Snippet: Fig. 1. TIMP-1fl/fl knockout mice develop a severe EAE phenotype that is immunologically independent. (A) Schematic of design of development of the flox-targeted sites that enabled cell and tissue-specific deletion of TIMP-1 in GFAP-Cre-TIMP-1fl/fl mice. (B) Immunocytochemical validation of CRE expression in astrocytes from GFAP-Cre:TIMP-1fl/fl mice, and (C) physiological validation of TIMP-1 mRNA expression in primary astrocytes and absence of TIMP-1 mRNA expression in astrocyte cultures from GFAP-Cre:TIMP-1fl/fl mice (n = 8 per treatment; ANOVA; ****P < 0.0001; where *P < 0.01; ***P < 0.002). (D) Validation of absent TIMP-1 protein expression in primary astrocyte cultures from GFAP-Cre:Timp1fl/f mice (n = 3 biological replicates). (E) Clinical EAE in MOG35-55-treated C57Bl/6 WT and GFAP-Cre:TIMP-1fl/fl mice over 52 d time course (n = 13 to 17/treatment and genotype; 2-way ANOVA, P < 0.0001, where post-hoc multiple-comparisons identified significance at *P < 0.05 and **P < 0.01, as indicated). (F and G) Flow cytometry analysis of overall CD4+/IFNγ+ T cells from spinal cords of Wt and GFAP-Cre:TIMP- 1fl/fl mice at peak clinical EAE illness (day 18; n = 3 per genotype and treatment), including (H) analysis of CD4+/IFNγ+ T cells (t test, unpaired t test, P < 0.20).

Techniques Used: Knock-Out, Biomarker Discovery, Expressing, Flow Cytometry

Fig. 2. Proteomic identification of elevated fibronectin (Fn) in secretome of TIMP-1KO astrocytes. (A and B) Topographic projections rendered using ProteomeLab™ of PF-2D analyses of extracellular proteins in secretome of primary astrocytes from WT (A and C) and TIMP-1KO (B and D) cultures. (C and D) Magnified view of differential peaks of interest identified by ProteomeLab™ which were interrogated by Liquid chromatography–mass spectrometry. (E) Western blot analysis of fibronectin (Fn) protein expression in ACM CM from WT and TIMP-1KO astrocytes. (F) Quantification of Fn from ACM from primary WT and TIMP-1KO astrocyte cultures showed TIMP-1KO astrocyte cultures had elevated Fn protein levels that were reduced with the addition of rmTIMP-1. (G) Analysis of Fn mRNA expression by qPCR demonstrated no TIMP-1 regulation of Fn mRNA expression in primary TIMP-1KO astrocyte cultures. (H) Validation of immunodepletion of Fn from ACM from primary WT and TIMP-1KO astrocyte cultures. (I) Effect of immunodepletion of Fn from ACM on OL differentiation in vitro in TIMP-1KO and Wt (control) treated cultures. Data in F–I were analyzed by ANOVA with either Bonferroni or Fisher’s least significant difference (LSD) post-hoc tests where appropriate *P < 0.05, **P < 0.01 as indicated. n = 3 biological replicates representing 3 to 4 averaged technical replicates/biological replicate.

Figure Legend Snippet: Fig. 2. Proteomic identification of elevated fibronectin (Fn) in secretome of TIMP-1KO astrocytes. (A and B) Topographic projections rendered using ProteomeLab™ of PF-2D analyses of extracellular proteins in secretome of primary astrocytes from WT (A and C) and TIMP-1KO (B and D) cultures. (C and D) Magnified view of differential peaks of interest identified by ProteomeLab™ which were interrogated by Liquid chromatography–mass spectrometry. (E) Western blot analysis of fibronectin (Fn) protein expression in ACM CM from WT and TIMP-1KO astrocytes. (F) Quantification of Fn from ACM from primary WT and TIMP-1KO astrocyte cultures showed TIMP-1KO astrocyte cultures had elevated Fn protein levels that were reduced with the addition of rmTIMP-1. (G) Analysis of Fn mRNA expression by qPCR demonstrated no TIMP-1 regulation of Fn mRNA expression in primary TIMP-1KO astrocyte cultures. (H) Validation of immunodepletion of Fn from ACM from primary WT and TIMP-1KO astrocyte cultures. (I) Effect of immunodepletion of Fn from ACM on OL differentiation in vitro in TIMP-1KO and Wt (control) treated cultures. Data in F–I were analyzed by ANOVA with either Bonferroni or Fisher’s least significant difference (LSD) post-hoc tests where appropriate *P < 0.05, **P < 0.01 as indicated. n = 3 biological replicates representing 3 to 4 averaged technical replicates/biological replicate.

Techniques Used: Liquid Chromatography, Mass Spectrometry, Western Blot, Expressing, Biomarker Discovery, Immunodepletion, In Vitro, Control

Fig. 5. Characterization of Fn and TIMP-1 expression in primary human astrocytes and identification of Ana in human MS proteomic databases. (A) Schematic representation of two modes of analysis of human astrocytes using direct ex vivo, (e.g., scRNAseq) which has indicated no expression or no changes in TIMP-1 expression in astrocytes in the MS brain, and, as used in the present study, in vitro cultures from patient donors. (B) Relative FN1, TIMP1 and ratio FN1 to TIMP1 mRNA expression in control human (ctrl) vs. MS astrocytes as analyzed by qRT-PCR. (C) FN, TIMP1, and ratio FN to TIMP1 protein expression in ctrl human vs. MS astrocytes as analyzed by western blot. (D) Expression of Fn protein in ctrl human and MS astrocytes after TIMP-1 treatment (10 ng/mL human rTIMP-1 for 48 h). The dotted line indicates no treatment (untreated), which was set to 1. * indicates significant difference from untreated control (P < 0.05), one sample t test); ** indicates a significant difference between the two groups (P < 0.01, unpaired student t test). Non-demented control (blue shades 3 to 6) and MS (orange shades, 3 to 6) donors are consistently color-coded in B–D. (E) Proteomic analysis reveals Fn and Ana in the CSF of MS patients. “Low” indicates low cytokine levels in the CSF, while “High” indicates high cytokine levels in the CSF. A and B represent technical replicates.

Figure Legend Snippet: Fig. 5. Characterization of Fn and TIMP-1 expression in primary human astrocytes and identification of Ana in human MS proteomic databases. (A) Schematic representation of two modes of analysis of human astrocytes using direct ex vivo, (e.g., scRNAseq) which has indicated no expression or no changes in TIMP-1 expression in astrocytes in the MS brain, and, as used in the present study, in vitro cultures from patient donors. (B) Relative FN1, TIMP1 and ratio FN1 to TIMP1 mRNA expression in control human (ctrl) vs. MS astrocytes as analyzed by qRT-PCR. (C) FN, TIMP1, and ratio FN to TIMP1 protein expression in ctrl human vs. MS astrocytes as analyzed by western blot. (D) Expression of Fn protein in ctrl human and MS astrocytes after TIMP-1 treatment (10 ng/mL human rTIMP-1 for 48 h). The dotted line indicates no treatment (untreated), which was set to 1. * indicates significant difference from untreated control (P < 0.05), one sample t test); ** indicates a significant difference between the two groups (P < 0.01, unpaired student t test). Non-demented control (blue shades 3 to 6) and MS (orange shades, 3 to 6) donors are consistently color-coded in B–D. (E) Proteomic analysis reveals Fn and Ana in the CSF of MS patients. “Low” indicates low cytokine levels in the CSF, while “High” indicates high cytokine levels in the CSF. A and B represent technical replicates.

Techniques Used: Expressing, Ex Vivo, In Vitro, Control, Quantitative RT-PCR, Western Blot

Fig. 6. Proposed model for astrocytic TIMP-1 regulation of OPC differentiation by Ana. Astrocytes when stimulated express and release TIMP-1, which under non-disease conditions would limit the production of Ana from fibronectin. In the absence of astrocytic TIMP-1, like which can occur under chronic inflammatory conditions, the elevated production of Ana impairs OPCs’ differentiation while also blocking the ability of FTY720 to promote differentiation. Together, these factors culminate in reduced remyelination of axons contributing to chronic demyelinating plaques in the CNS of MS patients.

Figure Legend Snippet: Fig. 6. Proposed model for astrocytic TIMP-1 regulation of OPC differentiation by Ana. Astrocytes when stimulated express and release TIMP-1, which under non-disease conditions would limit the production of Ana from fibronectin. In the absence of astrocytic TIMP-1, like which can occur under chronic inflammatory conditions, the elevated production of Ana impairs OPCs’ differentiation while also blocking the ability of FTY720 to promote differentiation. Together, these factors culminate in reduced remyelination of axons contributing to chronic demyelinating plaques in the CNS of MS patients.

Techniques Used: Blocking Assay

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Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Astrocytic TIMP-1 regulates production of Anastellin, an inhibitor of oligodendrocyte differentiation and FTY720 responses.

doi: 10.1073/pnas.2306816121

Figure Lengend Snippet: Fig. 1. TIMP-1fl/fl knockout mice develop a severe EAE phenotype that is immunologically independent. (A) Schematic of design of development of the flox-targeted sites that enabled cell and tissue-specific deletion of TIMP-1 in GFAP-Cre-TIMP-1fl/fl mice. (B) Immunocytochemical validation of CRE expression in astrocytes from GFAP-Cre:TIMP-1fl/fl mice, and (C) physiological validation of TIMP-1 mRNA expression in primary astrocytes and absence of TIMP-1 mRNA expression in astrocyte cultures from GFAP-Cre:TIMP-1fl/fl mice (n = 8 per treatment; ANOVA; ****P < 0.0001; where *P < 0.01; ***P < 0.002). (D) Validation of absent TIMP-1 protein expression in primary astrocyte cultures from GFAP-Cre:Timp1fl/f mice (n = 3 biological replicates). (E) Clinical EAE in MOG35-55-treated C57Bl/6 WT and GFAP-Cre:TIMP-1fl/fl mice over 52 d time course (n = 13 to 17/treatment and genotype; 2-way ANOVA, P < 0.0001, where post-hoc multiple-comparisons identified significance at *P < 0.05 and **P < 0.01, as indicated). (F and G) Flow cytometry analysis of overall CD4+/IFNγ+ T cells from spinal cords of Wt and GFAP-Cre:TIMP- 1fl/fl mice at peak clinical EAE illness (day 18; n = 3 per genotype and treatment), including (H) analysis of CD4+/IFNγ+ T cells (t test, unpaired t test, P < 0.20).

Article Snippet: Anti- Fibronectin antibody (Biotin) (ab6584) (Abcam; rabbit, 1:5,000), Anti- Fibronectin antibody (ab23750) (Abcam; rabbit, 1:100- immunodepletion), Anti- Fibronectin antibody (ab2033) (Abcam; rabbit, 1:200), Anti- Timp1 antibody (AF980) (R&D Systems; goat, 1:125), Anti- MBP antibody (12) (Millipore; rat monoclonal, 1:500), Mouse anti- A2B5 (Invitrogen, Carlsbad, CA; 3 μg/mL), DAPI (Invitrogen, 1:1,000), Mouse anti- GFAP (Millipore, 1:400), Beta- actin (Sigma Aldrich, St. Louis, MO; mouse, 1:10,000).

Techniques: Knock-Out, Biomarker Discovery, Expressing, Flow Cytometry

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Astrocytic TIMP-1 regulates production of Anastellin, an inhibitor of oligodendrocyte differentiation and FTY720 responses.

doi: 10.1073/pnas.2306816121

Figure Lengend Snippet: Fig. 2. Proteomic identification of elevated fibronectin (Fn) in secretome of TIMP-1KO astrocytes. (A and B) Topographic projections rendered using ProteomeLab™ of PF-2D analyses of extracellular proteins in secretome of primary astrocytes from WT (A and C) and TIMP-1KO (B and D) cultures. (C and D) Magnified view of differential peaks of interest identified by ProteomeLab™ which were interrogated by Liquid chromatography–mass spectrometry. (E) Western blot analysis of fibronectin (Fn) protein expression in ACM CM from WT and TIMP-1KO astrocytes. (F) Quantification of Fn from ACM from primary WT and TIMP-1KO astrocyte cultures showed TIMP-1KO astrocyte cultures had elevated Fn protein levels that were reduced with the addition of rmTIMP-1. (G) Analysis of Fn mRNA expression by qPCR demonstrated no TIMP-1 regulation of Fn mRNA expression in primary TIMP-1KO astrocyte cultures. (H) Validation of immunodepletion of Fn from ACM from primary WT and TIMP-1KO astrocyte cultures. (I) Effect of immunodepletion of Fn from ACM on OL differentiation in vitro in TIMP-1KO and Wt (control) treated cultures. Data in F–I were analyzed by ANOVA with either Bonferroni or Fisher’s least significant difference (LSD) post-hoc tests where appropriate *P < 0.05, **P < 0.01 as indicated. n = 3 biological replicates representing 3 to 4 averaged technical replicates/biological replicate.

Techniques: Liquid Chromatography, Mass Spectrometry, Western Blot, Expressing, Biomarker Discovery, Immunodepletion, In Vitro, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Astrocytic TIMP-1 regulates production of Anastellin, an inhibitor of oligodendrocyte differentiation and FTY720 responses.

doi: 10.1073/pnas.2306816121

Figure Lengend Snippet: Fig. 5. Characterization of Fn and TIMP-1 expression in primary human astrocytes and identification of Ana in human MS proteomic databases. (A) Schematic representation of two modes of analysis of human astrocytes using direct ex vivo, (e.g., scRNAseq) which has indicated no expression or no changes in TIMP-1 expression in astrocytes in the MS brain, and, as used in the present study, in vitro cultures from patient donors. (B) Relative FN1, TIMP1 and ratio FN1 to TIMP1 mRNA expression in control human (ctrl) vs. MS astrocytes as analyzed by qRT-PCR. (C) FN, TIMP1, and ratio FN to TIMP1 protein expression in ctrl human vs. MS astrocytes as analyzed by western blot. (D) Expression of Fn protein in ctrl human and MS astrocytes after TIMP-1 treatment (10 ng/mL human rTIMP-1 for 48 h). The dotted line indicates no treatment (untreated), which was set to 1. * indicates significant difference from untreated control (P < 0.05), one sample t test); ** indicates a significant difference between the two groups (P < 0.01, unpaired student t test). Non-demented control (blue shades 3 to 6) and MS (orange shades, 3 to 6) donors are consistently color-coded in B–D. (E) Proteomic analysis reveals Fn and Ana in the CSF of MS patients. “Low” indicates low cytokine levels in the CSF, while “High” indicates high cytokine levels in the CSF. A and B represent technical replicates.

Techniques: Expressing, Ex Vivo, In Vitro, Control, Quantitative RT-PCR, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Astrocytic TIMP-1 regulates production of Anastellin, an inhibitor of oligodendrocyte differentiation and FTY720 responses.

doi: 10.1073/pnas.2306816121

Figure Lengend Snippet: Fig. 6. Proposed model for astrocytic TIMP-1 regulation of OPC differentiation by Ana. Astrocytes when stimulated express and release TIMP-1, which under non-disease conditions would limit the production of Ana from fibronectin. In the absence of astrocytic TIMP-1, like which can occur under chronic inflammatory conditions, the elevated production of Ana impairs OPCs’ differentiation while also blocking the ability of FTY720 to promote differentiation. Together, these factors culminate in reduced remyelination of axons contributing to chronic demyelinating plaques in the CNS of MS patients.

Techniques: Blocking Assay

(A) Predicted inputs to TIMP1 in the PrP GRN, coloured as described in left panel of . (B) Representative immunohistochemistry image for αSMA (blue) and TIMP1 (brown) in non-plaque human aorta (N=7), scale bar=500 µm (overview) and 100 µm (zoomed view). (C) Enrichment plots from gene set enrichment analysis (GSEA) for E2F targets (p adj = 0.005) and G2M checkpoint genes (p adj = 0.04) in bulk RNA-seq data from human VSMCs (hVSMCs) treated with 500 ng/mL recombinant human (rh) TIMP1 for 6 hours versus control cells (N=6 independent hVSMC isolates). (D) Proportion of EdU+ hVSMCs following 16 hours EdU incorporation in cells treated with 500 ng/mL rhTIMP1 or vehicle controls (CTRL), standardised to the average of all samples (N=6 independent hVSMC isolates). (E) Representative images of Myh11-Confetti VSMCs isolated from murine aortas and cultured for 21 days in the presence of vehicle control, 500 ng/mL recombinant murine TIMP1 or 2 ng/mL PDGF-BB. Scale bar=500 µm. (F, G) Quantification of number and size of clonally expanded patches of Confetti+ VSMCs over 21 days of culture. Statistical significance assessed via a generalised linear model, N=4 animals.

Journal: bioRxiv

Article Title: Network-based prioritisation and validation of novel regulators of vascular smooth muscle cell proliferation in disease

doi: 10.1101/2023.08.25.554834

Figure Lengend Snippet: (A) Predicted inputs to TIMP1 in the PrP GRN, coloured as described in left panel of . (B) Representative immunohistochemistry image for αSMA (blue) and TIMP1 (brown) in non-plaque human aorta (N=7), scale bar=500 µm (overview) and 100 µm (zoomed view). (C) Enrichment plots from gene set enrichment analysis (GSEA) for E2F targets (p adj = 0.005) and G2M checkpoint genes (p adj = 0.04) in bulk RNA-seq data from human VSMCs (hVSMCs) treated with 500 ng/mL recombinant human (rh) TIMP1 for 6 hours versus control cells (N=6 independent hVSMC isolates). (D) Proportion of EdU+ hVSMCs following 16 hours EdU incorporation in cells treated with 500 ng/mL rhTIMP1 or vehicle controls (CTRL), standardised to the average of all samples (N=6 independent hVSMC isolates). (E) Representative images of Myh11-Confetti VSMCs isolated from murine aortas and cultured for 21 days in the presence of vehicle control, 500 ng/mL recombinant murine TIMP1 or 2 ng/mL PDGF-BB. Scale bar=500 µm. (F, G) Quantification of number and size of clonally expanded patches of Confetti+ VSMCs over 21 days of culture. Statistical significance assessed via a generalised linear model, N=4 animals.

Article Snippet: TIMP1-containing fractions were identified by Western blot analysis using the anti-TIMP1 antibody (human: rabbit anti-human antibody; 1:1,000, cat. #8946; Cell Signaling Technology, mouse: goat anti-murine antibody 1:1,000, cat. #AF980; Biotechne R&D biosystems), pooled and concentrated by centrifugal filter (Amicon Ultra 15, 10 kD; Merck).

Techniques: Immunohistochemistry, RNA Sequencing Assay, Recombinant, Isolation, Cell Culture

(A) Proteome profiler phosphokinase array of human VSMCs (hVSMCs) following 15 minutes treatment of 500 ng/mL recombinant human (rh) TIMP1 or vehicle control. Quantification of relative spot intensity by densitometry. (B) Western blot of total, pS727 and pT705 STAT3 in serum starved hVSMCs analysed 0, 5 10, 15 or 30 minutes after rhTIMP1 treatment. (N=5 hVSMC isolates). (C) Quantification of relative band intensity, normalised to total STAT3 levels (N=5 hVSMC isolates), statistical significance determined using ANOVA. (D) ChIP qPCR analysis of STAT3 binding at STAT3 targets ( TWIST and JUN ) and a negative control ( AMICA1 ) gene promoters, in serum starved control hVSMCs and 15 minutes following 500 ng/mL rhTIMP1 treatment. Graph shows anti-STAT3 and control IgG precipitated DNA as a percent of input. N=3 hVSMC isolates. (E) Representative images of STAT3 and Ki67 staining in serum starved control hVSMCs and after 15 mins 500 ng/mL rhTIMP1 treatment (N=3 hVSMC isolates analysed in triplicate). Arrowheads indicate KI67+ cells. Scale bar=50 µm. (F) Quantification of relative fluorescence intensity of nuclear pSTAT (S727) staining in panel E, for KI67+ and KI67-cells. (G) Representative images of VSMCs isolated from Myh11-Confetti aortas cultured 21 days in +/− 500 ng rhTIMP1 and/or 10 uM TT101 (N=6). Scale bar=500 µm. (H) Number and size of clonally expanded patches formed by lineage labelled VSMCs from Myh11-Confetti aortas, treated as indicated and imaged over 21 days. Q uantified using ImageJ. Statistical significance assessed via generalised linear model. N=6. Asterisk indicates p<0.05.

Journal: bioRxiv

Article Title: Network-based prioritisation and validation of novel regulators of vascular smooth muscle cell proliferation in disease

doi: 10.1101/2023.08.25.554834

Figure Lengend Snippet: (A) Proteome profiler phosphokinase array of human VSMCs (hVSMCs) following 15 minutes treatment of 500 ng/mL recombinant human (rh) TIMP1 or vehicle control. Quantification of relative spot intensity by densitometry. (B) Western blot of total, pS727 and pT705 STAT3 in serum starved hVSMCs analysed 0, 5 10, 15 or 30 minutes after rhTIMP1 treatment. (N=5 hVSMC isolates). (C) Quantification of relative band intensity, normalised to total STAT3 levels (N=5 hVSMC isolates), statistical significance determined using ANOVA. (D) ChIP qPCR analysis of STAT3 binding at STAT3 targets ( TWIST and JUN ) and a negative control ( AMICA1 ) gene promoters, in serum starved control hVSMCs and 15 minutes following 500 ng/mL rhTIMP1 treatment. Graph shows anti-STAT3 and control IgG precipitated DNA as a percent of input. N=3 hVSMC isolates. (E) Representative images of STAT3 and Ki67 staining in serum starved control hVSMCs and after 15 mins 500 ng/mL rhTIMP1 treatment (N=3 hVSMC isolates analysed in triplicate). Arrowheads indicate KI67+ cells. Scale bar=50 µm. (F) Quantification of relative fluorescence intensity of nuclear pSTAT (S727) staining in panel E, for KI67+ and KI67-cells. (G) Representative images of VSMCs isolated from Myh11-Confetti aortas cultured 21 days in +/− 500 ng rhTIMP1 and/or 10 uM TT101 (N=6). Scale bar=500 µm. (H) Number and size of clonally expanded patches formed by lineage labelled VSMCs from Myh11-Confetti aortas, treated as indicated and imaged over 21 days. Q uantified using ImageJ. Statistical significance assessed via generalised linear model. N=6. Asterisk indicates p<0.05.

Techniques: Recombinant, Western Blot, Binding Assay, Negative Control, Staining, Fluorescence, Isolation, Cell Culture

(A) Number and size of clonally expanded patches formed by lineage labelled VSMCs from Myh11-Confetti animals, treated with 500 ng/mL recombinant TIMP1 or equimolar amount of N-TIMP1 tracked over 21 days of culture. Statistical significance assessed via generalised linear model. N=4. (B) Representative image of CD74 immunostaining (magenta) in lineage labelled mouse carotids 10 days post ligation of Myh11-Confetti animals. Magnified panels also show DAPI counterstained nuclei (top: merge of Confetti signals, CD74 and DAPI, middle: DAPI and RFP, lower: DAPI and CD74). White pointers marks CD74/RFP double positive cells. N=5 animals. Scale bar=100 µm (overview), 30 µm (zoomed view) (C) Representative image of murine VSMCs treated with 500 ng/mL recombinant murine (rm) TIMP1 four days post isolation and immunostained for pSTAT3 (S727, red) and CD74 (green). Nuclei are counterstained with DAPI (blue). Red pointer marks a STAT3 high cells, arrowhead points to a STAT3 low cell in merged and individual channels. (D) Quantification of cellular CD74 levels in panel C, stratified by high or low nuclear pSTAT3 intensity. N=4 animals, 4 repeats of each. Statistical significance was determined via student t-test. (E) Number and size of clonally expanded patches formed by lineage labelled VSMCs from Myh11-Confetti animals, treated with 500 ng/mL rmTIMP1 and/or a CD74 blocking antibody or peptide. (F) Representative western blot of serum started control human VSMCs (hVSMCs, UT), and cells treated for 5 minutes with 500 ng/mL recombinant human TIMP1 with or without +/− pre-treatment with an antibody (CD74 ab) or a peptide (CD74 pep) that blocks CD74. ( G ) Densitometric quantification of panel F. N=4 hVSMC isolates. ( H ) Representative immunohistochemistry image of CD74 in non-plaque human aorta. αSMA = blue, CD74 = brown. Scale bar=500 µm (overview) and 100 µm (zoomed view). N=7. (I) Representative images of RNA in situ hybridization for ACTA2 (green), TIMP1 (blue) and CD74 (red) in healthy human aorta, and plaque containing carotid. Blue arrows denote TIMP1 / ACTA2 + cells, red arrows denote CD74 / ACTA2 +cells. ( J ) Quantification of the % of ACTA2 positive cells also expressing TIMP1 or CD74 in the medial layer of non-plaque aorta (Healthy media) or in medial (Media Under Plaque) or Plaque regions of carotid endarterectomy samples (N=4 for each condition).

Journal: bioRxiv

Article Title: Network-based prioritisation and validation of novel regulators of vascular smooth muscle cell proliferation in disease

doi: 10.1101/2023.08.25.554834

Figure Lengend Snippet: (A) Number and size of clonally expanded patches formed by lineage labelled VSMCs from Myh11-Confetti animals, treated with 500 ng/mL recombinant TIMP1 or equimolar amount of N-TIMP1 tracked over 21 days of culture. Statistical significance assessed via generalised linear model. N=4. (B) Representative image of CD74 immunostaining (magenta) in lineage labelled mouse carotids 10 days post ligation of Myh11-Confetti animals. Magnified panels also show DAPI counterstained nuclei (top: merge of Confetti signals, CD74 and DAPI, middle: DAPI and RFP, lower: DAPI and CD74). White pointers marks CD74/RFP double positive cells. N=5 animals. Scale bar=100 µm (overview), 30 µm (zoomed view) (C) Representative image of murine VSMCs treated with 500 ng/mL recombinant murine (rm) TIMP1 four days post isolation and immunostained for pSTAT3 (S727, red) and CD74 (green). Nuclei are counterstained with DAPI (blue). Red pointer marks a STAT3 high cells, arrowhead points to a STAT3 low cell in merged and individual channels. (D) Quantification of cellular CD74 levels in panel C, stratified by high or low nuclear pSTAT3 intensity. N=4 animals, 4 repeats of each. Statistical significance was determined via student t-test. (E) Number and size of clonally expanded patches formed by lineage labelled VSMCs from Myh11-Confetti animals, treated with 500 ng/mL rmTIMP1 and/or a CD74 blocking antibody or peptide. (F) Representative western blot of serum started control human VSMCs (hVSMCs, UT), and cells treated for 5 minutes with 500 ng/mL recombinant human TIMP1 with or without +/− pre-treatment with an antibody (CD74 ab) or a peptide (CD74 pep) that blocks CD74. ( G ) Densitometric quantification of panel F. N=4 hVSMC isolates. ( H ) Representative immunohistochemistry image of CD74 in non-plaque human aorta. αSMA = blue, CD74 = brown. Scale bar=500 µm (overview) and 100 µm (zoomed view). N=7. (I) Representative images of RNA in situ hybridization for ACTA2 (green), TIMP1 (blue) and CD74 (red) in healthy human aorta, and plaque containing carotid. Blue arrows denote TIMP1 / ACTA2 + cells, red arrows denote CD74 / ACTA2 +cells. ( J ) Quantification of the % of ACTA2 positive cells also expressing TIMP1 or CD74 in the medial layer of non-plaque aorta (Healthy media) or in medial (Media Under Plaque) or Plaque regions of carotid endarterectomy samples (N=4 for each condition).

Techniques: Recombinant, Immunostaining, Ligation, Isolation, Blocking Assay, Western Blot, Immunohistochemistry, RNA In Situ Hybridization, Expressing

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