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R&D Systems recombinant murine wisp1

Figure 1. WISP2 inhibits <t>WISP1-induced</t> collagen linearization. A, Scanning electron microscopy of Col I lattices formed in the presence of PBS (Col I), 50 mg/mL recombinant WISP1, 100 mg/mL recombinant WISP2, or 50 mg/mL WISP1 þ 100 mg/mL WISP2 (1:3 WISP1:WISP2 molar ratio). Magenta arrows, examples of “knot-like” structures; yellow arrows, examples of “hairpin- or end-like structures.” Scale bars, 2 mm. B, Curvature ratios of Col I ﬁbrils in lattices from A (n ¼ 15; three independent experiments; ﬁve images/lattice). C, Binding of WISP1 to ﬁbrillar Col I (or BSA as negative control) as determined by solid-phase binding assay and detected with an anti-WISP1 antibody (n ¼ 6, from three independent experiments). D, Binding of WISP2 to ﬁbrillar Col I (or BSA as negative control) as determined by solid-phase binding assay and detected with an anti-WISP2 antibody (n ¼ 4, from two independent experiments). E, Inhibition of WISP1 (1 mmol/L) binding to ﬁbrillar Col I in the presence of increasing concentrations of WISP2 (n ¼ 6, from three independent experiments). F, Binding of prebound WISP1 (1 mmol/L) to ﬁbrillar Col I in the presence of increasing concentrations of WISP2 (n ¼ 6, from three independent experiments). G, Binding of prebound WISP2 (2 mmol/L) to ﬁbrillar Col I in the presence of increasing concentrations of WISP1 (n ¼ 4, from two independent experiments). H, Binding of WISP1 to WISP2 (or BSA as negative control) as determined by solid-phase binding assay (n ¼ 4, from two independent experiments). Mean SEM (B–H); one-way ANOVA followed by Tukey posttest (B). ns, nonsigniﬁcant, P > 0.05; , P < 0.001.

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1) Product Images from "Matricellular Protein WISP2 Is an Endogenous Inhibitor of Collagen Linearization and Cancer Metastasis"

Article Title: Matricellular Protein WISP2 Is an Endogenous Inhibitor of Collagen Linearization and Cancer Metastasis

Journal: Cancer Research

doi: 10.1158/0008-5472.can-20-3982

Figure 1. WISP2 inhibits WISP1-induced collagen linearization. A, Scanning electron microscopy of Col I lattices formed in the presence of PBS (Col I), 50 mg/mL recombinant WISP1, 100 mg/mL recombinant WISP2, or 50 mg/mL WISP1 þ 100 mg/mL WISP2 (1:3 WISP1:WISP2 molar ratio). Magenta arrows, examples of “knot-like” structures; yellow arrows, examples of “hairpin- or end-like structures.” Scale bars, 2 mm. B, Curvature ratios of Col I ﬁbrils in lattices from A (n ¼ 15; three independent experiments; ﬁve images/lattice). C, Binding of WISP1 to ﬁbrillar Col I (or BSA as negative control) as determined by solid-phase binding assay and detected with an anti-WISP1 antibody (n ¼ 6, from three independent experiments). D, Binding of WISP2 to ﬁbrillar Col I (or BSA as negative control) as determined by solid-phase binding assay and detected with an anti-WISP2 antibody (n ¼ 4, from two independent experiments). E, Inhibition of WISP1 (1 mmol/L) binding to ﬁbrillar Col I in the presence of increasing concentrations of WISP2 (n ¼ 6, from three independent experiments). F, Binding of prebound WISP1 (1 mmol/L) to ﬁbrillar Col I in the presence of increasing concentrations of WISP2 (n ¼ 6, from three independent experiments). G, Binding of prebound WISP2 (2 mmol/L) to ﬁbrillar Col I in the presence of increasing concentrations of WISP1 (n ¼ 4, from two independent experiments). H, Binding of WISP1 to WISP2 (or BSA as negative control) as determined by solid-phase binding assay (n ¼ 4, from two independent experiments). Mean SEM (B–H); one-way ANOVA followed by Tukey posttest (B). ns, nonsigniﬁcant, P > 0.05; , P < 0.001.

Figure Legend Snippet: Figure 1. WISP2 inhibits WISP1-induced collagen linearization. A, Scanning electron microscopy of Col I lattices formed in the presence of PBS (Col I), 50 mg/mL recombinant WISP1, 100 mg/mL recombinant WISP2, or 50 mg/mL WISP1 þ 100 mg/mL WISP2 (1:3 WISP1:WISP2 molar ratio). Magenta arrows, examples of “knot-like” structures; yellow arrows, examples of “hairpin- or end-like structures.” Scale bars, 2 mm. B, Curvature ratios of Col I ﬁbrils in lattices from A (n ¼ 15; three independent experiments; ﬁve images/lattice). C, Binding of WISP1 to ﬁbrillar Col I (or BSA as negative control) as determined by solid-phase binding assay and detected with an anti-WISP1 antibody (n ¼ 6, from three independent experiments). D, Binding of WISP2 to ﬁbrillar Col I (or BSA as negative control) as determined by solid-phase binding assay and detected with an anti-WISP2 antibody (n ¼ 4, from two independent experiments). E, Inhibition of WISP1 (1 mmol/L) binding to ﬁbrillar Col I in the presence of increasing concentrations of WISP2 (n ¼ 6, from three independent experiments). F, Binding of prebound WISP1 (1 mmol/L) to ﬁbrillar Col I in the presence of increasing concentrations of WISP2 (n ¼ 6, from three independent experiments). G, Binding of prebound WISP2 (2 mmol/L) to ﬁbrillar Col I in the presence of increasing concentrations of WISP1 (n ¼ 4, from two independent experiments). H, Binding of WISP1 to WISP2 (or BSA as negative control) as determined by solid-phase binding assay (n ¼ 4, from two independent experiments). Mean SEM (B–H); one-way ANOVA followed by Tukey posttest (B). ns, nonsigniﬁcant, P > 0.05; , P < 0.001.

Techniques Used: Electron Microscopy, Recombinant, Binding Assay, Negative Control, Inhibition

Figure 2. WISP2 inhibits WISP1-induced cell invasion through Col I. A–C, WISP2 (A) or WISP1 (B) gene expression levels, or WISP2/WISP1 gene expression ratio (C) in tumors and adjacent normal tissues from patients with different cancer types (BRCA, breast invasive carcinoma, n ¼ 113 normal and n ¼ 1,119 tumors; COAD, colon adenocarcinoma, n ¼ 41 normal and n ¼ 483 tumors; LUAD, lung adenocarcinoma, n ¼ 59 normal and n ¼ 541 tumors; LUSC, lung squamous cell carcinoma, n ¼ 51 normal and n ¼ 502 tumors; PRAD, prostate adenocarcinoma, n ¼ 52 normal and n ¼ 502 tumors; READ, rectum adenocarcinoma, n ¼ 10 normal and n ¼ 167 tumors). D, Migratory tracks of 4T1 cells plated on Col I (n ¼ 72 cells), Col I þ WISP1 (n ¼ 77 cells), Col I þ WISP2 (n ¼ 72 cells), or Col I þ WISP1 þ WISP2 (n ¼ 62 cells) lattices. E, Average cell speed from cells tracked in D. F, Migratory tracks of 4T1 cells plated on plastic in presence of PBS vehicle control (n ¼ 69 cells), WISP1 (n ¼ 85 cells), WISP2 (n ¼ 81 cells), or WISP1 þ WISP2 (n ¼ 65 cells). G, Average cell speed from cells tracked in F. H, Invasion of 4T1 cells through Col I layered on Transwell inserts in the presence of WISP1, WISP2, or WISP1 þ WISP2 (1:3 WISP1:WISP2 molar ratio; n ¼ 6 biological replicates). I, Migration of 4T1 cells through the microporous membrane of Transwell inserts (in the absence of Col I) in the presence of PBS (vehicle), WISP1, WISP2, or WISP1 þ WISP2 (n ¼ 6 biological replicates). J, Proliferation rate of 4T1 cells plated on Col I, Col I þ WISP1, or Col I þ WISP2 lattices (n ¼ 4 biological replicates). K, Invasion of MDA-MB-231 cells through Col I layered on Transwell inserts in the presence of WISP1, WISP2, or WISP1 þ WISP2 (n ¼ 6 biological replicates). L, Migration of MDA-MB-231 cells through the microporous membrane of Transwell inserts (in the absence of Col I) in the presence of PBS (vehicle), WISP1, WISP2, or WISP1 þ WISP2 (n ¼ 6 biological replicates). M, Proliferation rate of MDA-MB-231 cells plated on Col I, Col I þ WISP1, or Col I þ WISP2 lattices (n ¼ 4 biological replicates). Mean SEM (A–C, E, and G–M); Mann–Whitney test (A–C) and one-way ANOVA followed by Tukey posttest (E, G–I, K, and L). ns, nonsigniﬁcant, P > 0.05; , P < 0.001.

Figure Legend Snippet: Figure 2. WISP2 inhibits WISP1-induced cell invasion through Col I. A–C, WISP2 (A) or WISP1 (B) gene expression levels, or WISP2/WISP1 gene expression ratio (C) in tumors and adjacent normal tissues from patients with different cancer types (BRCA, breast invasive carcinoma, n ¼ 113 normal and n ¼ 1,119 tumors; COAD, colon adenocarcinoma, n ¼ 41 normal and n ¼ 483 tumors; LUAD, lung adenocarcinoma, n ¼ 59 normal and n ¼ 541 tumors; LUSC, lung squamous cell carcinoma, n ¼ 51 normal and n ¼ 502 tumors; PRAD, prostate adenocarcinoma, n ¼ 52 normal and n ¼ 502 tumors; READ, rectum adenocarcinoma, n ¼ 10 normal and n ¼ 167 tumors). D, Migratory tracks of 4T1 cells plated on Col I (n ¼ 72 cells), Col I þ WISP1 (n ¼ 77 cells), Col I þ WISP2 (n ¼ 72 cells), or Col I þ WISP1 þ WISP2 (n ¼ 62 cells) lattices. E, Average cell speed from cells tracked in D. F, Migratory tracks of 4T1 cells plated on plastic in presence of PBS vehicle control (n ¼ 69 cells), WISP1 (n ¼ 85 cells), WISP2 (n ¼ 81 cells), or WISP1 þ WISP2 (n ¼ 65 cells). G, Average cell speed from cells tracked in F. H, Invasion of 4T1 cells through Col I layered on Transwell inserts in the presence of WISP1, WISP2, or WISP1 þ WISP2 (1:3 WISP1:WISP2 molar ratio; n ¼ 6 biological replicates). I, Migration of 4T1 cells through the microporous membrane of Transwell inserts (in the absence of Col I) in the presence of PBS (vehicle), WISP1, WISP2, or WISP1 þ WISP2 (n ¼ 6 biological replicates). J, Proliferation rate of 4T1 cells plated on Col I, Col I þ WISP1, or Col I þ WISP2 lattices (n ¼ 4 biological replicates). K, Invasion of MDA-MB-231 cells through Col I layered on Transwell inserts in the presence of WISP1, WISP2, or WISP1 þ WISP2 (n ¼ 6 biological replicates). L, Migration of MDA-MB-231 cells through the microporous membrane of Transwell inserts (in the absence of Col I) in the presence of PBS (vehicle), WISP1, WISP2, or WISP1 þ WISP2 (n ¼ 6 biological replicates). M, Proliferation rate of MDA-MB-231 cells plated on Col I, Col I þ WISP1, or Col I þ WISP2 lattices (n ¼ 4 biological replicates). Mean SEM (A–C, E, and G–M); Mann–Whitney test (A–C) and one-way ANOVA followed by Tukey posttest (E, G–I, K, and L). ns, nonsigniﬁcant, P > 0.05; , P < 0.001.

Techniques Used: Gene Expression, Control, Migration, Membrane, MANN-WHITNEY

Figure 3. The CT domain of WISP1 drives WISP1-induced Col I linearization and cell invasion but is dispensable for WISP1-Col I binding. A, Scheme of WISP constructs stably transduced in 4T1 cells. IGFBP, insulin-like growth factor domain; VWC, von Willebrand factor type C repeat domain; TSP-1, thrombospondin type-1 repeat domain; CT, cysteine-rich knot-like C-terminal domain. B, Detection of WISP1, WISP1DCT, WISP2, and WISP2þCT binding to ﬁbrillar Col I, using conditioned media from 4T1 cells stably overexpressing these constructs or stably transduced with an empty vector (EV) control. Col I-bound proteins were detected with anti-WISP1 (left) or anti-WISP2 (right) antibodies. Binding was normalized to the molarity of each construct in the conditioned medium (left, n ¼ 9, from three independent experiments; right, n ¼ 8, from four independent experiments). C, Binding of WISP1-myc protein to ﬁbrillar Col I in the presence of WISP1, WISP2, WISP1DCT, or WISP2þCT using 1:1 (v:v) ratios of conditioned medium from 4T1 cells overexpressing these proteins. Conditionedmedium from 4T1-EV (4T1 cells stably transduced with anempty vector) was used as negative control. Col I-bound proteins were detected with an anti-Myc antibody (n ¼ 4, from two independent experiments). D, Scanning electron microscopy of Col I lattices formed in the presence of concentrated conditioned medium from 4T1 stable cell lines. Scale bars, 2 mm. E, Curvature ratios of Col I ﬁbrils in lattices from D (n ¼ 15, three independent experiments, ﬁve images/lattice). Statistical signiﬁcance versus Col I þ EV is shown. F, Average cell speed of 4T1-EV (n ¼ 123), 4T1-Wisp1 (n ¼ 75), 4T1-Wisp2 (n ¼ 69), 4T1-Wisp1DCT (n ¼ 106), and 4T1-Wisp2þCT (n ¼ 66) cells plated on Col I lattices. G, Invasion of 4T1-EV, 4T1-Wisp1, 4T1-Wisp2,4T1-Wisp1DCT, or 4T1-WISP2þCT cells through ColI lattices layeredon Transwell inserts (n ¼ 9, except 4T1-EV and 4T1-Wisp1, n ¼ 15 biological replicates). H, Average cell speed of 4T1-EV (n ¼ 115), 4T1-Wisp1 (n ¼ 89), 4T1-Wisp2 (n ¼ 54), 4T1-Wisp1DCT (n ¼ 53), and 4T1-Wisp2þCT (n ¼ 59) cells plated on plastic. I, Migration of 4T1-EV, 4T1-Wisp1, 4T1-Wisp2, 4T1-Wisp1DCT, or 4T1-WISP2þCT cells through uncoated Transwell inserts (n ¼ 9, except 4T1-EV, n ¼ 18 biological replicates). B, C, and E–I, Mean SEM; one-way ANOVA followed by Tukey posttest. ns, nonsigniﬁcant, P > 0.05; , P < 0.01; , P < 0.001.

Figure Legend Snippet: Figure 3. The CT domain of WISP1 drives WISP1-induced Col I linearization and cell invasion but is dispensable for WISP1-Col I binding. A, Scheme of WISP constructs stably transduced in 4T1 cells. IGFBP, insulin-like growth factor domain; VWC, von Willebrand factor type C repeat domain; TSP-1, thrombospondin type-1 repeat domain; CT, cysteine-rich knot-like C-terminal domain. B, Detection of WISP1, WISP1DCT, WISP2, and WISP2þCT binding to ﬁbrillar Col I, using conditioned media from 4T1 cells stably overexpressing these constructs or stably transduced with an empty vector (EV) control. Col I-bound proteins were detected with anti-WISP1 (left) or anti-WISP2 (right) antibodies. Binding was normalized to the molarity of each construct in the conditioned medium (left, n ¼ 9, from three independent experiments; right, n ¼ 8, from four independent experiments). C, Binding of WISP1-myc protein to ﬁbrillar Col I in the presence of WISP1, WISP2, WISP1DCT, or WISP2þCT using 1:1 (v:v) ratios of conditioned medium from 4T1 cells overexpressing these proteins. Conditionedmedium from 4T1-EV (4T1 cells stably transduced with anempty vector) was used as negative control. Col I-bound proteins were detected with an anti-Myc antibody (n ¼ 4, from two independent experiments). D, Scanning electron microscopy of Col I lattices formed in the presence of concentrated conditioned medium from 4T1 stable cell lines. Scale bars, 2 mm. E, Curvature ratios of Col I ﬁbrils in lattices from D (n ¼ 15, three independent experiments, ﬁve images/lattice). Statistical signiﬁcance versus Col I þ EV is shown. F, Average cell speed of 4T1-EV (n ¼ 123), 4T1-Wisp1 (n ¼ 75), 4T1-Wisp2 (n ¼ 69), 4T1-Wisp1DCT (n ¼ 106), and 4T1-Wisp2þCT (n ¼ 66) cells plated on Col I lattices. G, Invasion of 4T1-EV, 4T1-Wisp1, 4T1-Wisp2,4T1-Wisp1DCT, or 4T1-WISP2þCT cells through ColI lattices layeredon Transwell inserts (n ¼ 9, except 4T1-EV and 4T1-Wisp1, n ¼ 15 biological replicates). H, Average cell speed of 4T1-EV (n ¼ 115), 4T1-Wisp1 (n ¼ 89), 4T1-Wisp2 (n ¼ 54), 4T1-Wisp1DCT (n ¼ 53), and 4T1-Wisp2þCT (n ¼ 59) cells plated on plastic. I, Migration of 4T1-EV, 4T1-Wisp1, 4T1-Wisp2, 4T1-Wisp1DCT, or 4T1-WISP2þCT cells through uncoated Transwell inserts (n ¼ 9, except 4T1-EV, n ¼ 18 biological replicates). B, C, and E–I, Mean SEM; one-way ANOVA followed by Tukey posttest. ns, nonsigniﬁcant, P > 0.05; , P < 0.01; , P < 0.001.

Techniques Used: Binding Assay, Construct, Stable Transfection, Transduction, Plasmid Preparation, Control, Negative Control, Electron Microscopy, Migration

Figure 4. WISP2 and WISP1DCT block TGFb1-induced cell invasion through Col I by acting as WISP1 antagonists. A, Relative Wisp1 mRNA expression levels in 4T1-EV (4T1 cells stably transduced with an empty vector), 4T1-Wisp1DCT, and 4T1-Wisp2 cells cultured in the presence of vehicle or TGFb1 (2 ng/mL). Primers targeting sequences coding in the CT domain of Wisp1 were used to detect endoge- nous Wisp1 expression levels but not overexpressed Wisp1DCT (n ¼ 3 biological replicates). B, Average cell speed of 4T1-EV þ vehicle (n ¼ 44), 4T1-EV þ 2 ng/mL TGFb1 (n ¼ 37), 4T1-Wisp2 þ vehicle (n ¼ 36), 4T1- Wisp2 cells þ TGFb1 (n ¼ 44), 4T1-Wisp1DCT þ vehicle (n ¼ 36), and 4T1-Wisp1DCT þ TGFb1 (n ¼ 35) cells plated on Col I lattices. C, Invasion of 4T1-EV, 4T1-Wisp2, and 4T1-Wisp1DCT cells treated with TGFb1 (2 ng/mL) or vehicle control through Col I lattices layered on Trans- well inserts (n ¼ 6 biological replicates). D, WISP1 protein concentration in conditioned medium from 4T1-gCont (4T1-indCas9 cells stably transduced with a pool of control nontargeting gRNAs) and 4T1-Wisp1KO (4T1 cells with CRISPR/Cas9 knockout of Wisp1) treated with TGFb1 (2 ng/mL) or vehicle control (n ¼ 4, from two independent experiments). E, Average cell speed of 4T1-gCont þ vehicle (n ¼ 72), 4T1-gCont þ TGFb1 (n ¼ 64), 4T1-Wisp1KO (n ¼ 68), 4T1-Wisp1KO þ TGFb1 (n ¼ 48) cells plated on Col I lattices. F, Invasion of 4T1-gCont and 4T1-Wisp1KO cells, treated with TGFb1 (2 ng/mL) or vehicle control, through Col I lattices layered on Transwell inserts (n ¼ 6 biological replicates). Mean SEM; two-way ANOVA followed by Tukey posttest. ns, nonsigniﬁcant, P > 0.05; , P < 0.05; , P < 0.01; , P < 0.001.

Figure Legend Snippet: Figure 4. WISP2 and WISP1DCT block TGFb1-induced cell invasion through Col I by acting as WISP1 antagonists. A, Relative Wisp1 mRNA expression levels in 4T1-EV (4T1 cells stably transduced with an empty vector), 4T1-Wisp1DCT, and 4T1-Wisp2 cells cultured in the presence of vehicle or TGFb1 (2 ng/mL). Primers targeting sequences coding in the CT domain of Wisp1 were used to detect endoge- nous Wisp1 expression levels but not overexpressed Wisp1DCT (n ¼ 3 biological replicates). B, Average cell speed of 4T1-EV þ vehicle (n ¼ 44), 4T1-EV þ 2 ng/mL TGFb1 (n ¼ 37), 4T1-Wisp2 þ vehicle (n ¼ 36), 4T1- Wisp2 cells þ TGFb1 (n ¼ 44), 4T1-Wisp1DCT þ vehicle (n ¼ 36), and 4T1-Wisp1DCT þ TGFb1 (n ¼ 35) cells plated on Col I lattices. C, Invasion of 4T1-EV, 4T1-Wisp2, and 4T1-Wisp1DCT cells treated with TGFb1 (2 ng/mL) or vehicle control through Col I lattices layered on Trans- well inserts (n ¼ 6 biological replicates). D, WISP1 protein concentration in conditioned medium from 4T1-gCont (4T1-indCas9 cells stably transduced with a pool of control nontargeting gRNAs) and 4T1-Wisp1KO (4T1 cells with CRISPR/Cas9 knockout of Wisp1) treated with TGFb1 (2 ng/mL) or vehicle control (n ¼ 4, from two independent experiments). E, Average cell speed of 4T1-gCont þ vehicle (n ¼ 72), 4T1-gCont þ TGFb1 (n ¼ 64), 4T1-Wisp1KO (n ¼ 68), 4T1-Wisp1KO þ TGFb1 (n ¼ 48) cells plated on Col I lattices. F, Invasion of 4T1-gCont and 4T1-Wisp1KO cells, treated with TGFb1 (2 ng/mL) or vehicle control, through Col I lattices layered on Transwell inserts (n ¼ 6 biological replicates). Mean SEM; two-way ANOVA followed by Tukey posttest. ns, nonsigniﬁcant, P > 0.05; , P < 0.05; , P < 0.01; , P < 0.001.

Techniques Used: Blocking Assay, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Cell Culture, Control, Protein Concentration, CRISPR, Knock-Out

Figure 5. WISP2 limits collagen linearization in tumors and inhibits breast cancer metastasis. A, Primary tumor mass, 28 days after orthotopic inoculation of 4T1-EV, 4T1-Wisp1, 4T1-Wisp2, or 4T1-Wisp1DCT cells into the 4th mammary fat pad of BALB/c female mice (n ¼ 10 mice/group, except 4T1-Wisp2, n ¼ 9 mice). B, Number of lung metastases in mice from A. C, Representative images of ﬁbrillar collagen in primary tumors from A visualized by Picrosirius red staining, followed by polarized light microscopy. Corresponding bright-ﬁeld images show tissue integ- rity. Scale bars, 200 mm. D, Average polarized light intensity in Picrosirius red staining images of tumors from A. E, Average stiffness (Young’s modulus) of tumors from A, measured by atomic force microsco- py. F, Scheme of experimental design. Recombinant WISP2 (rWISP2; 100 mg in 200 mL PBS) was admin- istered via intraperitoneal injection starting on day 8 post-injection of 4T1 cells. Tissues were collected 2 hours after the last dose of rWISP2. G, Primary tumor mass, 28 days after orthotopic inoculation of 4T1 cells into the 4th mammary fat pad (PBS, n ¼ 10; rWISP2, n ¼ 8 mice/group). H, Number of lung metastases in mice from G. I, Representative images of ﬁbrillar collagen in primary tumors from G visual- ized by Picrosirius red staining, followed by polarized light microscopy. Corresponding bright-ﬁeld images show tissue integrity. Scale bars, 200 mm. J, Aver- age polarized light intensity in Picrosirius red staining images of tumors from G. Mean SEM (A, B, D, E, G, H, and J); one-way ANOVA followed by Tukey posttest (A, B, D, and E). G, H, and J, Unpaired two-sided t test. ns, nonsigniﬁcant, P > 0.05; , P < 0.05; , P < 0.01, , P < 0.001.

Figure Legend Snippet: Figure 5. WISP2 limits collagen linearization in tumors and inhibits breast cancer metastasis. A, Primary tumor mass, 28 days after orthotopic inoculation of 4T1-EV, 4T1-Wisp1, 4T1-Wisp2, or 4T1-Wisp1DCT cells into the 4th mammary fat pad of BALB/c female mice (n ¼ 10 mice/group, except 4T1-Wisp2, n ¼ 9 mice). B, Number of lung metastases in mice from A. C, Representative images of ﬁbrillar collagen in primary tumors from A visualized by Picrosirius red staining, followed by polarized light microscopy. Corresponding bright-ﬁeld images show tissue integ- rity. Scale bars, 200 mm. D, Average polarized light intensity in Picrosirius red staining images of tumors from A. E, Average stiffness (Young’s modulus) of tumors from A, measured by atomic force microsco- py. F, Scheme of experimental design. Recombinant WISP2 (rWISP2; 100 mg in 200 mL PBS) was admin- istered via intraperitoneal injection starting on day 8 post-injection of 4T1 cells. Tissues were collected 2 hours after the last dose of rWISP2. G, Primary tumor mass, 28 days after orthotopic inoculation of 4T1 cells into the 4th mammary fat pad (PBS, n ¼ 10; rWISP2, n ¼ 8 mice/group). H, Number of lung metastases in mice from G. I, Representative images of ﬁbrillar collagen in primary tumors from G visual- ized by Picrosirius red staining, followed by polarized light microscopy. Corresponding bright-ﬁeld images show tissue integrity. Scale bars, 200 mm. J, Aver- age polarized light intensity in Picrosirius red staining images of tumors from G. Mean SEM (A, B, D, E, G, H, and J); one-way ANOVA followed by Tukey posttest (A, B, D, and E). G, H, and J, Unpaired two-sided t test. ns, nonsigniﬁcant, P > 0.05; , P < 0.05; , P < 0.01, , P < 0.001.

Techniques Used: Staining, Light Microscopy, Recombinant, Injection

Figure 6. WISP1 promotes whereas WISP2 inhi- bits human breast cancer metastasis. A, Invasion of MDA-MB-231 cells over- expressing WISP1 (MDA-WISP1) or WISP2 (MDA-WISP2), or stably trans- duced with an empty vector control (MDA-EV) through Col I lattices layered on Transwell inserts (n ¼ 9 biological replicates). B, Migration of MDA-EV, MDA-WISP1, or MDA-WISP2 cells through uncoated Transwell inserts (n ¼ 6 biological replicates). C, Prolifer- ation rate of MDA-EV, MDA-WISP1, or MDA-WISP2 cells plated on Col I lattices. (n ¼ 12 biological replicates). D, Primary tumor mass, 70 days after orthotopic inoculation of MDA-EV, MDA-WISP1, or MDA-WISP2 cells into the 4th mammary fat pad of NSG female mice (n ¼ 7 mice/ group). E, Number of lung metastases in mice from D. F, Representative images of ﬁbrillar collagen in primary tumors from D visualized by Picrosirius red staining, followed by polarized light microscopy. Corresponding bright- ﬁeld images show tissue integrity. Scale bars, 200 mm. G, Average polarized light intensity in Picrosirius red staining images of tumors from D. Mean SEM (A–E and G); one-way ANOVA followed by Tukey posttest (A, B, D, E, and G). ns, nonsigniﬁcant, P > 0.05; , P < 0.05; , P < 0.01; , P < 0.001.

Figure Legend Snippet: Figure 6. WISP1 promotes whereas WISP2 inhi- bits human breast cancer metastasis. A, Invasion of MDA-MB-231 cells over- expressing WISP1 (MDA-WISP1) or WISP2 (MDA-WISP2), or stably trans- duced with an empty vector control (MDA-EV) through Col I lattices layered on Transwell inserts (n ¼ 9 biological replicates). B, Migration of MDA-EV, MDA-WISP1, or MDA-WISP2 cells through uncoated Transwell inserts (n ¼ 6 biological replicates). C, Prolifer- ation rate of MDA-EV, MDA-WISP1, or MDA-WISP2 cells plated on Col I lattices. (n ¼ 12 biological replicates). D, Primary tumor mass, 70 days after orthotopic inoculation of MDA-EV, MDA-WISP1, or MDA-WISP2 cells into the 4th mammary fat pad of NSG female mice (n ¼ 7 mice/ group). E, Number of lung metastases in mice from D. F, Representative images of ﬁbrillar collagen in primary tumors from D visualized by Picrosirius red staining, followed by polarized light microscopy. Corresponding bright- ﬁeld images show tissue integrity. Scale bars, 200 mm. G, Average polarized light intensity in Picrosirius red staining images of tumors from D. Mean SEM (A–E and G); one-way ANOVA followed by Tukey posttest (A, B, D, E, and G). ns, nonsigniﬁcant, P > 0.05; , P < 0.05; , P < 0.01; , P < 0.001.

Techniques Used: Expressing, Stable Transfection, Plasmid Preparation, Control, Migration, Staining, Light Microscopy

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Journal: Cancer Research

Article Title: Matricellular Protein WISP2 Is an Endogenous Inhibitor of Collagen Linearization and Cancer Metastasis

doi: 10.1158/0008-5472.can-20-3982

Figure Lengend Snippet: Figure 1. WISP2 inhibits WISP1-induced collagen linearization. A, Scanning electron microscopy of Col I lattices formed in the presence of PBS (Col I), 50 mg/mL recombinant WISP1, 100 mg/mL recombinant WISP2, or 50 mg/mL WISP1 þ 100 mg/mL WISP2 (1:3 WISP1:WISP2 molar ratio). Magenta arrows, examples of “knot-like” structures; yellow arrows, examples of “hairpin- or end-like structures.” Scale bars, 2 mm. B, Curvature ratios of Col I ﬁbrils in lattices from A (n ¼ 15; three independent experiments; ﬁve images/lattice). C, Binding of WISP1 to ﬁbrillar Col I (or BSA as negative control) as determined by solid-phase binding assay and detected with an anti-WISP1 antibody (n ¼ 6, from three independent experiments). D, Binding of WISP2 to ﬁbrillar Col I (or BSA as negative control) as determined by solid-phase binding assay and detected with an anti-WISP2 antibody (n ¼ 4, from two independent experiments). E, Inhibition of WISP1 (1 mmol/L) binding to ﬁbrillar Col I in the presence of increasing concentrations of WISP2 (n ¼ 6, from three independent experiments). F, Binding of prebound WISP1 (1 mmol/L) to ﬁbrillar Col I in the presence of increasing concentrations of WISP2 (n ¼ 6, from three independent experiments). G, Binding of prebound WISP2 (2 mmol/L) to ﬁbrillar Col I in the presence of increasing concentrations of WISP1 (n ¼ 4, from two independent experiments). H, Binding of WISP1 to WISP2 (or BSA as negative control) as determined by solid-phase binding assay (n ¼ 4, from two independent experiments). Mean SEM (B–H); one-way ANOVA followed by Tukey posttest (B). ns, nonsigniﬁcant, P > 0.05; , P < 0.001.

Article Snippet: Recombinant murine WISP1 (R&D Systems, catalog no. 1680-WS) in PBS or recombinant human WISP2 (PeproTech, catalog no. 120-16) were added at a final concentration of 50 and 100 mg/mL, respectively, unless otherwise indicated.

Techniques: Electron Microscopy, Recombinant, Binding Assay, Negative Control, Inhibition

Journal: Cancer Research

Article Title: Matricellular Protein WISP2 Is an Endogenous Inhibitor of Collagen Linearization and Cancer Metastasis

doi: 10.1158/0008-5472.can-20-3982

Figure Lengend Snippet: Figure 2. WISP2 inhibits WISP1-induced cell invasion through Col I. A–C, WISP2 (A) or WISP1 (B) gene expression levels, or WISP2/WISP1 gene expression ratio (C) in tumors and adjacent normal tissues from patients with different cancer types (BRCA, breast invasive carcinoma, n ¼ 113 normal and n ¼ 1,119 tumors; COAD, colon adenocarcinoma, n ¼ 41 normal and n ¼ 483 tumors; LUAD, lung adenocarcinoma, n ¼ 59 normal and n ¼ 541 tumors; LUSC, lung squamous cell carcinoma, n ¼ 51 normal and n ¼ 502 tumors; PRAD, prostate adenocarcinoma, n ¼ 52 normal and n ¼ 502 tumors; READ, rectum adenocarcinoma, n ¼ 10 normal and n ¼ 167 tumors). D, Migratory tracks of 4T1 cells plated on Col I (n ¼ 72 cells), Col I þ WISP1 (n ¼ 77 cells), Col I þ WISP2 (n ¼ 72 cells), or Col I þ WISP1 þ WISP2 (n ¼ 62 cells) lattices. E, Average cell speed from cells tracked in D. F, Migratory tracks of 4T1 cells plated on plastic in presence of PBS vehicle control (n ¼ 69 cells), WISP1 (n ¼ 85 cells), WISP2 (n ¼ 81 cells), or WISP1 þ WISP2 (n ¼ 65 cells). G, Average cell speed from cells tracked in F. H, Invasion of 4T1 cells through Col I layered on Transwell inserts in the presence of WISP1, WISP2, or WISP1 þ WISP2 (1:3 WISP1:WISP2 molar ratio; n ¼ 6 biological replicates). I, Migration of 4T1 cells through the microporous membrane of Transwell inserts (in the absence of Col I) in the presence of PBS (vehicle), WISP1, WISP2, or WISP1 þ WISP2 (n ¼ 6 biological replicates). J, Proliferation rate of 4T1 cells plated on Col I, Col I þ WISP1, or Col I þ WISP2 lattices (n ¼ 4 biological replicates). K, Invasion of MDA-MB-231 cells through Col I layered on Transwell inserts in the presence of WISP1, WISP2, or WISP1 þ WISP2 (n ¼ 6 biological replicates). L, Migration of MDA-MB-231 cells through the microporous membrane of Transwell inserts (in the absence of Col I) in the presence of PBS (vehicle), WISP1, WISP2, or WISP1 þ WISP2 (n ¼ 6 biological replicates). M, Proliferation rate of MDA-MB-231 cells plated on Col I, Col I þ WISP1, or Col I þ WISP2 lattices (n ¼ 4 biological replicates). Mean SEM (A–C, E, and G–M); Mann–Whitney test (A–C) and one-way ANOVA followed by Tukey posttest (E, G–I, K, and L). ns, nonsigniﬁcant, P > 0.05; , P < 0.001.

Techniques: Gene Expression, Control, Migration, Membrane, MANN-WHITNEY

Journal: Cancer Research

Article Title: Matricellular Protein WISP2 Is an Endogenous Inhibitor of Collagen Linearization and Cancer Metastasis

doi: 10.1158/0008-5472.can-20-3982

Figure Lengend Snippet: Figure 3. The CT domain of WISP1 drives WISP1-induced Col I linearization and cell invasion but is dispensable for WISP1-Col I binding. A, Scheme of WISP constructs stably transduced in 4T1 cells. IGFBP, insulin-like growth factor domain; VWC, von Willebrand factor type C repeat domain; TSP-1, thrombospondin type-1 repeat domain; CT, cysteine-rich knot-like C-terminal domain. B, Detection of WISP1, WISP1DCT, WISP2, and WISP2þCT binding to ﬁbrillar Col I, using conditioned media from 4T1 cells stably overexpressing these constructs or stably transduced with an empty vector (EV) control. Col I-bound proteins were detected with anti-WISP1 (left) or anti-WISP2 (right) antibodies. Binding was normalized to the molarity of each construct in the conditioned medium (left, n ¼ 9, from three independent experiments; right, n ¼ 8, from four independent experiments). C, Binding of WISP1-myc protein to ﬁbrillar Col I in the presence of WISP1, WISP2, WISP1DCT, or WISP2þCT using 1:1 (v:v) ratios of conditioned medium from 4T1 cells overexpressing these proteins. Conditionedmedium from 4T1-EV (4T1 cells stably transduced with anempty vector) was used as negative control. Col I-bound proteins were detected with an anti-Myc antibody (n ¼ 4, from two independent experiments). D, Scanning electron microscopy of Col I lattices formed in the presence of concentrated conditioned medium from 4T1 stable cell lines. Scale bars, 2 mm. E, Curvature ratios of Col I ﬁbrils in lattices from D (n ¼ 15, three independent experiments, ﬁve images/lattice). Statistical signiﬁcance versus Col I þ EV is shown. F, Average cell speed of 4T1-EV (n ¼ 123), 4T1-Wisp1 (n ¼ 75), 4T1-Wisp2 (n ¼ 69), 4T1-Wisp1DCT (n ¼ 106), and 4T1-Wisp2þCT (n ¼ 66) cells plated on Col I lattices. G, Invasion of 4T1-EV, 4T1-Wisp1, 4T1-Wisp2,4T1-Wisp1DCT, or 4T1-WISP2þCT cells through ColI lattices layeredon Transwell inserts (n ¼ 9, except 4T1-EV and 4T1-Wisp1, n ¼ 15 biological replicates). H, Average cell speed of 4T1-EV (n ¼ 115), 4T1-Wisp1 (n ¼ 89), 4T1-Wisp2 (n ¼ 54), 4T1-Wisp1DCT (n ¼ 53), and 4T1-Wisp2þCT (n ¼ 59) cells plated on plastic. I, Migration of 4T1-EV, 4T1-Wisp1, 4T1-Wisp2, 4T1-Wisp1DCT, or 4T1-WISP2þCT cells through uncoated Transwell inserts (n ¼ 9, except 4T1-EV, n ¼ 18 biological replicates). B, C, and E–I, Mean SEM; one-way ANOVA followed by Tukey posttest. ns, nonsigniﬁcant, P > 0.05; , P < 0.01; , P < 0.001.

Techniques: Binding Assay, Construct, Stable Transfection, Transduction, Plasmid Preparation, Control, Negative Control, Electron Microscopy, Migration

Journal: Cancer Research

Article Title: Matricellular Protein WISP2 Is an Endogenous Inhibitor of Collagen Linearization and Cancer Metastasis

doi: 10.1158/0008-5472.can-20-3982

Figure Lengend Snippet: Figure 4. WISP2 and WISP1DCT block TGFb1-induced cell invasion through Col I by acting as WISP1 antagonists. A, Relative Wisp1 mRNA expression levels in 4T1-EV (4T1 cells stably transduced with an empty vector), 4T1-Wisp1DCT, and 4T1-Wisp2 cells cultured in the presence of vehicle or TGFb1 (2 ng/mL). Primers targeting sequences coding in the CT domain of Wisp1 were used to detect endoge- nous Wisp1 expression levels but not overexpressed Wisp1DCT (n ¼ 3 biological replicates). B, Average cell speed of 4T1-EV þ vehicle (n ¼ 44), 4T1-EV þ 2 ng/mL TGFb1 (n ¼ 37), 4T1-Wisp2 þ vehicle (n ¼ 36), 4T1- Wisp2 cells þ TGFb1 (n ¼ 44), 4T1-Wisp1DCT þ vehicle (n ¼ 36), and 4T1-Wisp1DCT þ TGFb1 (n ¼ 35) cells plated on Col I lattices. C, Invasion of 4T1-EV, 4T1-Wisp2, and 4T1-Wisp1DCT cells treated with TGFb1 (2 ng/mL) or vehicle control through Col I lattices layered on Trans- well inserts (n ¼ 6 biological replicates). D, WISP1 protein concentration in conditioned medium from 4T1-gCont (4T1-indCas9 cells stably transduced with a pool of control nontargeting gRNAs) and 4T1-Wisp1KO (4T1 cells with CRISPR/Cas9 knockout of Wisp1) treated with TGFb1 (2 ng/mL) or vehicle control (n ¼ 4, from two independent experiments). E, Average cell speed of 4T1-gCont þ vehicle (n ¼ 72), 4T1-gCont þ TGFb1 (n ¼ 64), 4T1-Wisp1KO (n ¼ 68), 4T1-Wisp1KO þ TGFb1 (n ¼ 48) cells plated on Col I lattices. F, Invasion of 4T1-gCont and 4T1-Wisp1KO cells, treated with TGFb1 (2 ng/mL) or vehicle control, through Col I lattices layered on Transwell inserts (n ¼ 6 biological replicates). Mean SEM; two-way ANOVA followed by Tukey posttest. ns, nonsigniﬁcant, P > 0.05; , P < 0.05; , P < 0.01; , P < 0.001.

Techniques: Blocking Assay, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Cell Culture, Control, Protein Concentration, CRISPR, Knock-Out

Journal: Cancer Research

Article Title: Matricellular Protein WISP2 Is an Endogenous Inhibitor of Collagen Linearization and Cancer Metastasis

doi: 10.1158/0008-5472.can-20-3982

Figure Lengend Snippet: Figure 5. WISP2 limits collagen linearization in tumors and inhibits breast cancer metastasis. A, Primary tumor mass, 28 days after orthotopic inoculation of 4T1-EV, 4T1-Wisp1, 4T1-Wisp2, or 4T1-Wisp1DCT cells into the 4th mammary fat pad of BALB/c female mice (n ¼ 10 mice/group, except 4T1-Wisp2, n ¼ 9 mice). B, Number of lung metastases in mice from A. C, Representative images of ﬁbrillar collagen in primary tumors from A visualized by Picrosirius red staining, followed by polarized light microscopy. Corresponding bright-ﬁeld images show tissue integ- rity. Scale bars, 200 mm. D, Average polarized light intensity in Picrosirius red staining images of tumors from A. E, Average stiffness (Young’s modulus) of tumors from A, measured by atomic force microsco- py. F, Scheme of experimental design. Recombinant WISP2 (rWISP2; 100 mg in 200 mL PBS) was admin- istered via intraperitoneal injection starting on day 8 post-injection of 4T1 cells. Tissues were collected 2 hours after the last dose of rWISP2. G, Primary tumor mass, 28 days after orthotopic inoculation of 4T1 cells into the 4th mammary fat pad (PBS, n ¼ 10; rWISP2, n ¼ 8 mice/group). H, Number of lung metastases in mice from G. I, Representative images of ﬁbrillar collagen in primary tumors from G visual- ized by Picrosirius red staining, followed by polarized light microscopy. Corresponding bright-ﬁeld images show tissue integrity. Scale bars, 200 mm. J, Aver- age polarized light intensity in Picrosirius red staining images of tumors from G. Mean SEM (A, B, D, E, G, H, and J); one-way ANOVA followed by Tukey posttest (A, B, D, and E). G, H, and J, Unpaired two-sided t test. ns, nonsigniﬁcant, P > 0.05; , P < 0.05; , P < 0.01, , P < 0.001.

Techniques: Staining, Light Microscopy, Recombinant, Injection

Journal: Cancer Research

Article Title: Matricellular Protein WISP2 Is an Endogenous Inhibitor of Collagen Linearization and Cancer Metastasis

doi: 10.1158/0008-5472.can-20-3982

Figure Lengend Snippet: Figure 6. WISP1 promotes whereas WISP2 inhi- bits human breast cancer metastasis. A, Invasion of MDA-MB-231 cells over- expressing WISP1 (MDA-WISP1) or WISP2 (MDA-WISP2), or stably trans- duced with an empty vector control (MDA-EV) through Col I lattices layered on Transwell inserts (n ¼ 9 biological replicates). B, Migration of MDA-EV, MDA-WISP1, or MDA-WISP2 cells through uncoated Transwell inserts (n ¼ 6 biological replicates). C, Prolifer- ation rate of MDA-EV, MDA-WISP1, or MDA-WISP2 cells plated on Col I lattices. (n ¼ 12 biological replicates). D, Primary tumor mass, 70 days after orthotopic inoculation of MDA-EV, MDA-WISP1, or MDA-WISP2 cells into the 4th mammary fat pad of NSG female mice (n ¼ 7 mice/ group). E, Number of lung metastases in mice from D. F, Representative images of ﬁbrillar collagen in primary tumors from D visualized by Picrosirius red staining, followed by polarized light microscopy. Corresponding bright- ﬁeld images show tissue integrity. Scale bars, 200 mm. G, Average polarized light intensity in Picrosirius red staining images of tumors from D. Mean SEM (A–E and G); one-way ANOVA followed by Tukey posttest (A, B, D, E, and G). ns, nonsigniﬁcant, P > 0.05; , P < 0.05; , P < 0.01; , P < 0.001.

Techniques: Expressing, Stable Transfection, Plasmid Preparation, Control, Migration, Staining, Light Microscopy

a Representative images of antibody arrays incubated with p14 or 20wo mouse serum. Spots corresponding to Wisp1 are marked with an empty square box. b Serum levels of Wisp1 in p14 ( n = 7), p28 ( n = 4), 11wo ( n = 6) and 20wo ( n = 7) mice measured with ELISA. c Plasma levels of WISP1 in children ( n = 11) and adult ( n = 14) measured with ELISA. d Quantification by qPCR of the expression of the indicated CCN genes in p14 ( n = 7, green) and 20wo mouse islets ( n = 6 for Cyr61 , n = 7 for other genes, gray). Values are expressed relative to Tbp . e Quantification by qPCR of the expression of the indicated CCN genes in adult human islets ( n = 6 for CYR61 , n = 5 for other genes). Values are expressed relative to TBP . f Quantification by qPCR of Wisp1 gene expression in the indicated p14 (green) and 20wo (gray) mouse tissues. Wisp1 gene expression is shown relative to levels in p14 bone, given the value of 1 ( n = 9 for bone, n = 4 for all other tissues). WAT: white adipose tissue; Gastroc: gastrocnemius. All data shown represent mean ± SEM from the indicated n . Indicated comparisons were made using two-tailed Student’s t test ( c , d ), one-way ( b ) and two-way ( f ) ANOVA. * p < 0.05; ** p < 0.01; **** p < 0.0001; ns: not significant. In f , at p14, bone Wisp1 gene expression was significantly higher than in all other tissues tested with p < 0.01–0.001.

Journal: Nature Communications

Article Title: Wisp1 is a circulating factor that stimulates proliferation of adult mouse and human beta cells

doi: 10.1038/s41467-020-19657-1

Figure Lengend Snippet: a Representative images of antibody arrays incubated with p14 or 20wo mouse serum. Spots corresponding to Wisp1 are marked with an empty square box. b Serum levels of Wisp1 in p14 ( n = 7), p28 ( n = 4), 11wo ( n = 6) and 20wo ( n = 7) mice measured with ELISA. c Plasma levels of WISP1 in children ( n = 11) and adult ( n = 14) measured with ELISA. d Quantification by qPCR of the expression of the indicated CCN genes in p14 ( n = 7, green) and 20wo mouse islets ( n = 6 for Cyr61 , n = 7 for other genes, gray). Values are expressed relative to Tbp . e Quantification by qPCR of the expression of the indicated CCN genes in adult human islets ( n = 6 for CYR61 , n = 5 for other genes). Values are expressed relative to TBP . f Quantification by qPCR of Wisp1 gene expression in the indicated p14 (green) and 20wo (gray) mouse tissues. Wisp1 gene expression is shown relative to levels in p14 bone, given the value of 1 ( n = 9 for bone, n = 4 for all other tissues). WAT: white adipose tissue; Gastroc: gastrocnemius. All data shown represent mean ± SEM from the indicated n . Indicated comparisons were made using two-tailed Student’s t test ( c , d ), one-way ( b ) and two-way ( f ) ANOVA. * p < 0.05; ** p < 0.01; **** p < 0.0001; ns: not significant. In f , at p14, bone Wisp1 gene expression was significantly higher than in all other tissues tested with p < 0.01–0.001.

Article Snippet: Recombinant mouse Wisp1 and human WISP1 proteins were purchased from R&D Systems.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Expressing, Two Tailed Test

a – c Beta cell proliferation in fixed pancreases from p14 Wisp1 + / + or Wisp1 − / − mice. a Representative images of pancreases co-immunostained for insulin (purple)/ki67 (green) or insulin (purple) /pHH3 (green). Nuclei are marked with Hoechst in blue. b Quantification of the percentage of beta (insulin+) cells that are ki67+ in Wisp1 + / + ( n = 5, yellow) or Wisp1 − / − ( n = 6, orange) mice. c Quantification of the percentage of beta (insulin+) cells that are pHH3+ in Wisp1 + / + ( n = 4, yellow) or Wisp1 − / − ( n = 5, orange) mice. d – f Beta cell proliferation in fixed pancreases from p12 Wisp1 − / − mice treated with saline or with rmWisp1 protein for three days (from p9 to p11). d Representative images of pancreases co-immunostained for insulin (purple)/ki67 (green) or insulin (purple)/pHH3 (green). Nuclei are marked with Hoechst in blue. e Quantification of the percentage of beta (insulin+) cells that are ki67+ in mice injected with rmWisp1 ( n = 4, orange) o saline ( n = 4, brown). f Quantification of the percentage of beta (insulin+) cells that are pHH3+ in mice injected with rmWisp1 ( n = 3, orange) o saline ( n = 3, brown). g – i Beta cell proliferation of 20wo mouse islet grafts transplanted into the anterior chamber of the eye of p16 Wisp1 + / + or Wisp1 − / − mouse recipients. g Representative images of islet grafts co-immunostained for insulin (purple)/ki67 (green) or insulin (purple)/pHH3 (green). Nuclei are marked with Hoechst in blue. h Quantification of the percentage of beta (insulin+) cells that are ki67+ in p16 Wisp1 + / + ( n = 7, yellow) or Wisp1 − / − ( n = 7, orange) mice. i Quantification of the percentage of beta (insulin+) cells that are pHH3+ in p16 Wisp1 + / + ( n = 4, yellow) or Wisp1 − / − ( n = 4, orange) mice. All data values represent mean ± SEM for the indicated n . * p < 0.05; ** p < 0.01 using two-tailed Student’s t test. Scale bars are 25 μm. ND: not detectable.

Journal: Nature Communications

Article Title: Wisp1 is a circulating factor that stimulates proliferation of adult mouse and human beta cells

doi: 10.1038/s41467-020-19657-1

Figure Lengend Snippet: a – c Beta cell proliferation in fixed pancreases from p14 Wisp1 + / + or Wisp1 − / − mice. a Representative images of pancreases co-immunostained for insulin (purple)/ki67 (green) or insulin (purple) /pHH3 (green). Nuclei are marked with Hoechst in blue. b Quantification of the percentage of beta (insulin+) cells that are ki67+ in Wisp1 + / + ( n = 5, yellow) or Wisp1 − / − ( n = 6, orange) mice. c Quantification of the percentage of beta (insulin+) cells that are pHH3+ in Wisp1 + / + ( n = 4, yellow) or Wisp1 − / − ( n = 5, orange) mice. d – f Beta cell proliferation in fixed pancreases from p12 Wisp1 − / − mice treated with saline or with rmWisp1 protein for three days (from p9 to p11). d Representative images of pancreases co-immunostained for insulin (purple)/ki67 (green) or insulin (purple)/pHH3 (green). Nuclei are marked with Hoechst in blue. e Quantification of the percentage of beta (insulin+) cells that are ki67+ in mice injected with rmWisp1 ( n = 4, orange) o saline ( n = 4, brown). f Quantification of the percentage of beta (insulin+) cells that are pHH3+ in mice injected with rmWisp1 ( n = 3, orange) o saline ( n = 3, brown). g – i Beta cell proliferation of 20wo mouse islet grafts transplanted into the anterior chamber of the eye of p16 Wisp1 + / + or Wisp1 − / − mouse recipients. g Representative images of islet grafts co-immunostained for insulin (purple)/ki67 (green) or insulin (purple)/pHH3 (green). Nuclei are marked with Hoechst in blue. h Quantification of the percentage of beta (insulin+) cells that are ki67+ in p16 Wisp1 + / + ( n = 7, yellow) or Wisp1 − / − ( n = 7, orange) mice. i Quantification of the percentage of beta (insulin+) cells that are pHH3+ in p16 Wisp1 + / + ( n = 4, yellow) or Wisp1 − / − ( n = 4, orange) mice. All data values represent mean ± SEM for the indicated n . * p < 0.05; ** p < 0.01 using two-tailed Student’s t test. Scale bars are 25 μm. ND: not detectable.

Article Snippet: Recombinant mouse Wisp1 and human WISP1 proteins were purchased from R&D Systems.

Techniques: Saline, Injection, Two Tailed Test

Adenoviruses encoding human WISP1 (Ad-WISP1) or beta-galactosidase (Ad-betaGal) were injected via the tail vein into 12wo C57BL6/J mice. a Quantification by qPCR of human WISP1 transcripts in the livers of mice seven days and fourteen days ( n = 4) post-injection. Levels are expressed relative to values in mice injected with Ad-betaGal, given the value of 1. b Quantification by qPCR of mouse Wisp1 mRNA ( n = 5 for Ad-betaGal, red; n = 7 for Ad-WISP1, purple) and human WISP1 transcripts ( n = 5 for Ad-betaGal, red; n = 4 for Ad-WISP1, purple) in the livers of mice fourteen days post-injection. Expression levels are expressed relative to Tbp . c Serum human WISP1 levels were measured by ELISA at days 7 and 14 post-injection of Ad-betaGal ( n = 7) or Ad-WISP1 ( n = 8 at day 7; n = 4 at day 14, purple). Human WISP1 was not detectable (ND) in serum from mice injected with Ad-betaGal. d , e Beta cell proliferation following injection of Ad-WISP1 and Ad-betaGal. d Representative images of in toto immunofluorescence staining against ki67 (green) and insulin (purple) in islets isolated at day 7 after injection of the indicated adenoviruses. Nuclei are labeled with Hoechst (blue). e Percentage of beta cells (insulin+) that are ki67+ at day 7 after injection of Ad-betaGal ( n = 4, red) or Ad-WISP1 ( n = 7, purple). f Beta cell mass at day 14 following injection of Ad-betaGal ( n = 5, red) or Ad-WISP1 ( n = 7, purple). All data shown represent mean ± SEM for the indicated n . * p < 0.05; ** p < 0.01 using two-tailed Student’s t test ( b , e , f ) or two-way ANOVA ( a , c ). Scale bars are 25 μm.

Journal: Nature Communications

Article Title: Wisp1 is a circulating factor that stimulates proliferation of adult mouse and human beta cells

doi: 10.1038/s41467-020-19657-1

Figure Lengend Snippet: Adenoviruses encoding human WISP1 (Ad-WISP1) or beta-galactosidase (Ad-betaGal) were injected via the tail vein into 12wo C57BL6/J mice. a Quantification by qPCR of human WISP1 transcripts in the livers of mice seven days and fourteen days ( n = 4) post-injection. Levels are expressed relative to values in mice injected with Ad-betaGal, given the value of 1. b Quantification by qPCR of mouse Wisp1 mRNA ( n = 5 for Ad-betaGal, red; n = 7 for Ad-WISP1, purple) and human WISP1 transcripts ( n = 5 for Ad-betaGal, red; n = 4 for Ad-WISP1, purple) in the livers of mice fourteen days post-injection. Expression levels are expressed relative to Tbp . c Serum human WISP1 levels were measured by ELISA at days 7 and 14 post-injection of Ad-betaGal ( n = 7) or Ad-WISP1 ( n = 8 at day 7; n = 4 at day 14, purple). Human WISP1 was not detectable (ND) in serum from mice injected with Ad-betaGal. d , e Beta cell proliferation following injection of Ad-WISP1 and Ad-betaGal. d Representative images of in toto immunofluorescence staining against ki67 (green) and insulin (purple) in islets isolated at day 7 after injection of the indicated adenoviruses. Nuclei are labeled with Hoechst (blue). e Percentage of beta cells (insulin+) that are ki67+ at day 7 after injection of Ad-betaGal ( n = 4, red) or Ad-WISP1 ( n = 7, purple). f Beta cell mass at day 14 following injection of Ad-betaGal ( n = 5, red) or Ad-WISP1 ( n = 7, purple). All data shown represent mean ± SEM for the indicated n . * p < 0.05; ** p < 0.01 using two-tailed Student’s t test ( b , e , f ) or two-way ANOVA ( a , c ). Scale bars are 25 μm.

Article Snippet: Recombinant mouse Wisp1 and human WISP1 proteins were purchased from R&D Systems.

Techniques: Injection, Expressing, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Isolation, Labeling, Two Tailed Test

$a Schematic of experimental plan. b Quantification by qPCR of mouse Wisp1 mRNA ( n = 6 for Ad-betaGal, yellow; n = 10 for Ad-WISP1, green) and human WISP1 transcripts ( n = 8 for Ad-betaGal, yellow; n = 6 for Ad-WISP1, green) in the livers of mice fourteen days post-injection. Expression levels are expressed relative to Tbp . c Serum human WISP1 levels were measured by ELISA at days 9 and 14 post-injection with Ad-betaGal ( n = 5) or Ad-WISP1 ( n = 4, green). Human WISP1 was not detectable (ND) in mice injected with Ad-betaGal. d Blood glucose concentrations measured at the indicated days post-injection with Ad-betaGal ( n = 8, yellow) or Ad-WISP1 ( n = 7, green). e Serum insulin at day 14 following administration of Ad-betaGal ( n = 11, yellow) or Ad-WISP1 ( n = 13, green). f , g Beta cell proliferation following injection of Ad-WISP1 and Ad-betaGal. f Representative images of immunofluorescence staining against ki67 (green) and insulin (purple) in fixed pancreases at day 14 after injection of the indicated adenoviruses. Nuclei are labeled with Hoechst (blue). g Percentage of beta cells (insulin+) that are ki67+ at day 14 days after injection of the indicated adenoviruses ( n = 7; Ad-betaGal in yellow, Ad-WISP1 in green). h Beta cell fractional area (insulin+ area relative to total pancreatic area) and i total beta cell mass at day 14 after injection of Ad-betaGal ( n = 8, yellow) or Ad-WISP1 ( n = 7, green). All data shown represent mean ± SEM for the indicated n . * p < 0.05; ** p < 0.01 using two-tailed Student’s t test ( b , e) , one-tailed Student’s t test ( g – i ) and two-way ANOVA ( d ). Scale bars are 25 μm.$

Journal: Nature Communications

Article Title: Wisp1 is a circulating factor that stimulates proliferation of adult mouse and human beta cells

doi: 10.1038/s41467-020-19657-1

Figure Lengend Snippet: a Schematic of experimental plan. b Quantification by qPCR of mouse Wisp1 mRNA ( n = 6 for Ad-betaGal, yellow; n = 10 for Ad-WISP1, green) and human WISP1 transcripts ( n = 8 for Ad-betaGal, yellow; n = 6 for Ad-WISP1, green) in the livers of mice fourteen days post-injection. Expression levels are expressed relative to Tbp . c Serum human WISP1 levels were measured by ELISA at days 9 and 14 post-injection with Ad-betaGal ( n = 5) or Ad-WISP1 ( n = 4, green). Human WISP1 was not detectable (ND) in mice injected with Ad-betaGal. d Blood glucose concentrations measured at the indicated days post-injection with Ad-betaGal ( n = 8, yellow) or Ad-WISP1 ( n = 7, green). e Serum insulin at day 14 following administration of Ad-betaGal ( n = 11, yellow) or Ad-WISP1 ( n = 13, green). f , g Beta cell proliferation following injection of Ad-WISP1 and Ad-betaGal. f Representative images of immunofluorescence staining against ki67 (green) and insulin (purple) in fixed pancreases at day 14 after injection of the indicated adenoviruses. Nuclei are labeled with Hoechst (blue). g Percentage of beta cells (insulin+) that are ki67+ at day 14 days after injection of the indicated adenoviruses ( n = 7; Ad-betaGal in yellow, Ad-WISP1 in green). h Beta cell fractional area (insulin+ area relative to total pancreatic area) and i total beta cell mass at day 14 after injection of Ad-betaGal ( n = 8, yellow) or Ad-WISP1 ( n = 7, green). All data shown represent mean ± SEM for the indicated n . * p < 0.05; ** p < 0.01 using two-tailed Student’s t test ( b , e) , one-tailed Student’s t test ( g – i ) and two-way ANOVA ( d ). Scale bars are 25 μm.

Article Snippet: Recombinant mouse Wisp1 and human WISP1 proteins were purchased from R&D Systems.

Techniques: Injection, Expressing, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Labeling, Two Tailed Test, One-tailed Test

a , b Beta cell proliferation in mouse islets incubated with Wisp1 recombinant mouse protein (rmWisp1). a Representative images of in toto immunofluorescence showing ki67 (green) and insulin (purple) staining in mouse islets cultured for 48 h with increasing amounts of rmWisp1 protein or harmine. Nuclei are marked with Hoechst in blue. b Percentage of beta cells (insulin+) that are ki67+ in cultured islets under the indicated conditions (control, n = 49 islets, in gray; rmWisp1-250, n = 19 islets, in blue; rmWisp1-500, n = 45 islets, in green; harmine, n = 15 islets, in pink; from four independent experiments except for harmine that are from two independent experiments). c , d Beta cell proliferation in mouse islets co-cultured with NIH3T3 cells expressing WISP1. c Representative images of in toto immunofluorescence showing ki67 (green) and insulin (purple) staining in mouse islets co-cultured for 24 or 48 h with NIH3T3 cells infected with the indicated adenoviruses. Nuclei are marked with Hoechst in blue. d Percentage of beta cells (insulin+) that are ki67+ in mouse islets cultured under the indicated conditions for 24 h (control, in gray: n = 25 islets; 3T3/WISP1, in blue: n = 21 islets, from three independent experiments) and for 48 h (control, in gray: n = 100 islets; 3T3/WISP1, in blue: n = 88 islets, from three independent experiments). e , f Beta cell proliferation in human islets incubated with recombinant human WISP1 protein (rhWISP1). e Representative images of in toto immunofluorescence showing ki67 (green) and insulin (purple) staining in human islets incubated for 48 h with increasing amounts of rhWISP1 protein or with harmine. Nuclei are marked with Hoechst in blue. f Percentage of beta cells (insulin+) that are ki67+ in human islets cultured under the indicated conditions (control, in gray: n = 79 islets; rhWISP1-250, in blue: n = 60 islets; rhWISP1-500, in green: n = 98 islets; harmine, in pink: n = 17 islets; from four donors). All data shown represent mean ± SEM for the indicated n . Comparisons were made using one-way ANOVA. * p < 0.05; ** p < 0.01; **** p < 0.0001. Scale bars are 25 μm.

Journal: Nature Communications

Article Title: Wisp1 is a circulating factor that stimulates proliferation of adult mouse and human beta cells

doi: 10.1038/s41467-020-19657-1

Figure Lengend Snippet: a , b Beta cell proliferation in mouse islets incubated with Wisp1 recombinant mouse protein (rmWisp1). a Representative images of in toto immunofluorescence showing ki67 (green) and insulin (purple) staining in mouse islets cultured for 48 h with increasing amounts of rmWisp1 protein or harmine. Nuclei are marked with Hoechst in blue. b Percentage of beta cells (insulin+) that are ki67+ in cultured islets under the indicated conditions (control, n = 49 islets, in gray; rmWisp1-250, n = 19 islets, in blue; rmWisp1-500, n = 45 islets, in green; harmine, n = 15 islets, in pink; from four independent experiments except for harmine that are from two independent experiments). c , d Beta cell proliferation in mouse islets co-cultured with NIH3T3 cells expressing WISP1. c Representative images of in toto immunofluorescence showing ki67 (green) and insulin (purple) staining in mouse islets co-cultured for 24 or 48 h with NIH3T3 cells infected with the indicated adenoviruses. Nuclei are marked with Hoechst in blue. d Percentage of beta cells (insulin+) that are ki67+ in mouse islets cultured under the indicated conditions for 24 h (control, in gray: n = 25 islets; 3T3/WISP1, in blue: n = 21 islets, from three independent experiments) and for 48 h (control, in gray: n = 100 islets; 3T3/WISP1, in blue: n = 88 islets, from three independent experiments). e , f Beta cell proliferation in human islets incubated with recombinant human WISP1 protein (rhWISP1). e Representative images of in toto immunofluorescence showing ki67 (green) and insulin (purple) staining in human islets incubated for 48 h with increasing amounts of rhWISP1 protein or with harmine. Nuclei are marked with Hoechst in blue. f Percentage of beta cells (insulin+) that are ki67+ in human islets cultured under the indicated conditions (control, in gray: n = 79 islets; rhWISP1-250, in blue: n = 60 islets; rhWISP1-500, in green: n = 98 islets; harmine, in pink: n = 17 islets; from four donors). All data shown represent mean ± SEM for the indicated n . Comparisons were made using one-way ANOVA. * p < 0.05; ** p < 0.01; **** p < 0.0001. Scale bars are 25 μm.

Article Snippet: Recombinant mouse Wisp1 and human WISP1 proteins were purchased from R&D Systems.

Techniques: Incubation, Recombinant, Immunofluorescence, Staining, Cell Culture, Expressing, Infection

a Determination of Akt activation ( Ser473 phosphorylation) by immunoblot analysis in mouse islets incubated with recombinant mouse Wisp1 protein (rmWisp1) at 500 ng/ml for 30 min. Top: representative immunoblot image. Molecular weight markers are shown on the right. Bottom, quantification of Akt activation, expressed relative to control islets (no rmWisp1), given the value of 1 (control, in gray: n = 10; rmWisp1, in blue: n = 11, from five independent experiments). b , c Beta cell proliferation in mouse islets incubated with rmWisp1 protein and Akt inhibitors. b Representative immunofluorescence images showing ki67 staining in green and insulin in purple. Nuclei are marked with Hoechst (blue). c Percentage of beta cells (insulin+) that are ki67+ in islets incubated with rmWisp1protein at 500 ng/ml for 48 h alone ( n = 41 islets, blue) or with the Akt inhibitors AZD5363 ( n = 21 islets, pink) or Akti ( n = 25 islets, yellow), or left untreated (control, gray: n = 39 islets) from three different isolation experiments. d Determination of AKT activation ( Ser473 phosphorylation) by immunoblot analysis in human islets incubated with recombinant human WISP1 protein (rhWISP1) at 500 ng/ml for 15 min. Top: representative immunoblot image. Molecular weight markers are shown on the right. Bottom, quantification of AKT activation, expressed relative to control islets (no WISP1), given the value of 1 (control in gray: n = 12; rhWISP1 in blue: n = 7, from 3 donors) e , f Beta cell proliferation in human islets incubated with Wisp1 protein and Akt inhibitors. e Representative immunofluorescence images showing ki67 staining in green and insulin in red. Nuclei are marked with Hoechst (blue). f Percentage of beta cells (ins+) that are ki67+ in human islets incubated with rhWISP1 protein at 500 ng/ml for 48 h alone ( n = 21 islets, blue) or with the Akt inhibitors AZD5363 ( n = 31 islets, pink) or Akti ( n = 15 islets, yellow), or left untreated (control, gray: n = 20 islets) from 3 donors. All data shown represent mean ± SEM for the indicated n . Comparisons were made using two-tailed Student’s t test ( a , d ) or one-way ANOVA ( c , f ). * p < 0.05; *** p < 0.001; **** p < 0.0001. Scale bars are 25 μm.

Journal: Nature Communications

Article Title: Wisp1 is a circulating factor that stimulates proliferation of adult mouse and human beta cells

doi: 10.1038/s41467-020-19657-1

Figure Lengend Snippet: a Determination of Akt activation ( Ser473 phosphorylation) by immunoblot analysis in mouse islets incubated with recombinant mouse Wisp1 protein (rmWisp1) at 500 ng/ml for 30 min. Top: representative immunoblot image. Molecular weight markers are shown on the right. Bottom, quantification of Akt activation, expressed relative to control islets (no rmWisp1), given the value of 1 (control, in gray: n = 10; rmWisp1, in blue: n = 11, from five independent experiments). b , c Beta cell proliferation in mouse islets incubated with rmWisp1 protein and Akt inhibitors. b Representative immunofluorescence images showing ki67 staining in green and insulin in purple. Nuclei are marked with Hoechst (blue). c Percentage of beta cells (insulin+) that are ki67+ in islets incubated with rmWisp1protein at 500 ng/ml for 48 h alone ( n = 41 islets, blue) or with the Akt inhibitors AZD5363 ( n = 21 islets, pink) or Akti ( n = 25 islets, yellow), or left untreated (control, gray: n = 39 islets) from three different isolation experiments. d Determination of AKT activation ( Ser473 phosphorylation) by immunoblot analysis in human islets incubated with recombinant human WISP1 protein (rhWISP1) at 500 ng/ml for 15 min. Top: representative immunoblot image. Molecular weight markers are shown on the right. Bottom, quantification of AKT activation, expressed relative to control islets (no WISP1), given the value of 1 (control in gray: n = 12; rhWISP1 in blue: n = 7, from 3 donors) e , f Beta cell proliferation in human islets incubated with Wisp1 protein and Akt inhibitors. e Representative immunofluorescence images showing ki67 staining in green and insulin in red. Nuclei are marked with Hoechst (blue). f Percentage of beta cells (ins+) that are ki67+ in human islets incubated with rhWISP1 protein at 500 ng/ml for 48 h alone ( n = 21 islets, blue) or with the Akt inhibitors AZD5363 ( n = 31 islets, pink) or Akti ( n = 15 islets, yellow), or left untreated (control, gray: n = 20 islets) from 3 donors. All data shown represent mean ± SEM for the indicated n . Comparisons were made using two-tailed Student’s t test ( a , d ) or one-way ANOVA ( c , f ). * p < 0.05; *** p < 0.001; **** p < 0.0001. Scale bars are 25 μm.

Article Snippet: Recombinant mouse Wisp1 and human WISP1 proteins were purchased from R&D Systems.

Techniques: Activation Assay, Western Blot, Incubation, Recombinant, Molecular Weight, Immunofluorescence, Staining, Isolation, Two Tailed Test

(A) Pie chart of transcripts detected by genome-wide profiling that are upregulated in FAPs isolated from young muscles at 3 dpi compared to the uninjured condition and classified as secreted proteins. (B) Venn diagram of genes induced in FAPs isolated from young muscles at 3 dpi compared to the uninjured condition (light grey), differentially regulated between FAPs isolated from young and aged muscles at 3 dpi (purple), and encoding proteins annotated as “signaling molecules” by the Panther database (dark grey). (C) WISP1 mRNA levels measured by qPCR in FAPs isolated from young and aged muscles under uninjured conditions or at 3 dpi. (D) WISP1 mRNA levels measured by qPCR in muscles from young and aged mice under uninjured conditions or at 3, 7 and 14 dpi (n=8 mice per condition). (E) WISP1 protein levels in regenerating muscles from young and aged mice under uninjured conditions or at 3, 7 and 14 dpi. n≥5 mice per condition. Arbitrary units (A.U.). (A-C) n≥5 replicates per condition, with cells pooled from multiple mice for each. (C-E) Data are represented as means ± S.E.M. p-values are *p<0.05, **p<0.01, ***p<0.001 using an ANOVA followed by a Bonferroni post hoc test. See also Figures S4 and S5.

Journal: Cell stem cell

Article Title: Aging Disrupts Muscle Stem Cell Function by Impairing Matricellular WISP1 Secretion from Fibro-Adipogenic Progenitors

doi: 10.1016/j.stem.2018.12.014

Figure Lengend Snippet: (A) Pie chart of transcripts detected by genome-wide profiling that are upregulated in FAPs isolated from young muscles at 3 dpi compared to the uninjured condition and classified as secreted proteins. (B) Venn diagram of genes induced in FAPs isolated from young muscles at 3 dpi compared to the uninjured condition (light grey), differentially regulated between FAPs isolated from young and aged muscles at 3 dpi (purple), and encoding proteins annotated as “signaling molecules” by the Panther database (dark grey). (C) WISP1 mRNA levels measured by qPCR in FAPs isolated from young and aged muscles under uninjured conditions or at 3 dpi. (D) WISP1 mRNA levels measured by qPCR in muscles from young and aged mice under uninjured conditions or at 3, 7 and 14 dpi (n=8 mice per condition). (E) WISP1 protein levels in regenerating muscles from young and aged mice under uninjured conditions or at 3, 7 and 14 dpi. n≥5 mice per condition. Arbitrary units (A.U.). (A-C) n≥5 replicates per condition, with cells pooled from multiple mice for each. (C-E) Data are represented as means ± S.E.M. p-values are *p<0.05, **p<0.01, ***p<0.001 using an ANOVA followed by a Bonferroni post hoc test. See also Figures S4 and S5.

Article Snippet: For in vivo WISP1 treatment, mouse recombinant WISP1 protein (R&D # 1680-WS) was administered daily by intra-peritoneal injections at 1mg/kg per day during the course of muscle regeneration after injury.

Techniques: Genome Wide, Isolation

(A) Quantification of the number of Td+ MuSCs from young uninjured muscles relative to co-cultured FAPs from wild-type (WT) or WISP1 knockout (WISP1−/−) muscles for 12 h after isolation. n=24 replicates per condition, repeated twice with cells from different mice for each condition. (B-D) qPCR quantification of Pax7, MyoD and Myogenin (MyoG) mRNA from WT and WISP1−/− muscles under uninjured (uninj.) conditions or at 3 and 7 dpi. (E) Representative Pax7 and MyoD immunostaining of WT and WISP1−/− muscle cross sections at 3 dpi. White arrowheads indicate Pax7+/MyoD+ MuSCs. Scale bar = 100 μm. (F) Quantification of the number of Pax7+/MyoD+ MuSCs in WT and WISP1−/− muscle cross sections at 3 dpi. (G) Representative Laminin immunostaining of WT and WISP1−/− muscle cross sections at 14 dpi. Scale bar = 100 μm. (H) Quantification of the cross-sectional area distribution of regenerating fibers with centralized nuclei in WT and WISP1−/− muscles at 14 dpi. (B-D, F and H) n≥5 mice per condition. (A, B-D, F and H) Data are represented as means ± S.E.M. p-values are *p<0.05, **p<0.01, ***p<0.001, #p<0.1 using a Mann-Whitney test when comparing two conditions, and ANOVA followed by a Bonferroni post hoc test when comparing multiple conditions, and a Kolmogorov-Smirnov test to assess fiber cross-sectional area distributions. See also Figure S6.

Journal: Cell stem cell

Article Title: Aging Disrupts Muscle Stem Cell Function by Impairing Matricellular WISP1 Secretion from Fibro-Adipogenic Progenitors

doi: 10.1016/j.stem.2018.12.014

Figure Lengend Snippet: (A) Quantification of the number of Td+ MuSCs from young uninjured muscles relative to co-cultured FAPs from wild-type (WT) or WISP1 knockout (WISP1−/−) muscles for 12 h after isolation. n=24 replicates per condition, repeated twice with cells from different mice for each condition. (B-D) qPCR quantification of Pax7, MyoD and Myogenin (MyoG) mRNA from WT and WISP1−/− muscles under uninjured (uninj.) conditions or at 3 and 7 dpi. (E) Representative Pax7 and MyoD immunostaining of WT and WISP1−/− muscle cross sections at 3 dpi. White arrowheads indicate Pax7+/MyoD+ MuSCs. Scale bar = 100 μm. (F) Quantification of the number of Pax7+/MyoD+ MuSCs in WT and WISP1−/− muscle cross sections at 3 dpi. (G) Representative Laminin immunostaining of WT and WISP1−/− muscle cross sections at 14 dpi. Scale bar = 100 μm. (H) Quantification of the cross-sectional area distribution of regenerating fibers with centralized nuclei in WT and WISP1−/− muscles at 14 dpi. (B-D, F and H) n≥5 mice per condition. (A, B-D, F and H) Data are represented as means ± S.E.M. p-values are *p<0.05, **p<0.01, ***p<0.001, #p<0.1 using a Mann-Whitney test when comparing two conditions, and ANOVA followed by a Bonferroni post hoc test when comparing multiple conditions, and a Kolmogorov-Smirnov test to assess fiber cross-sectional area distributions. See also Figure S6.

Techniques: Cell Culture, Knock-Out, Isolation, Immunostaining, MANN-WHITNEY

(A) Quantification of the number of MuSCs cultured for 36 h after isolation from uninjured muscles of aged mice in media containing vehicle (Veh.) or 8 μg/ml WISP1. (B) Quantification of the number of EdU+ MuSCs cultured for 3 days after isolation from uninjured muscles of aged mice in media containing vehicle (Veh.) or WISP1. (C) Representative images of EdU+ MuSCs cultured for 3 days after isolation from uninjured muscles of aged mice in media containing vehicle (Veh.) or WISP1. Scale bars = 50 μm. (D) Pax7 and YFP immunostaining of an asymmetric MuSC division on a single myofiber from a Myf5-Cre/R26R-YFP mouse. Scale bar = 25 μm (top), 5 μm (bottom). (E) Quantification of the number of symmetric (YFP−/YFP−) and asymmetric (YFP−/YFP+) divisions per single myofiber cultured in media containing Veh. or WISP1 for 42 h after isolation from adult uninjured EDL muscles of Myf5-Cre/R26R-YFP mice. n=3 mice per condition, n≥30 fibers analyzed per mouse. (F and G) Primary myoblasts were treated with either Veh. or WISP1 for 24 h and phospho-Akt (p-Akt) and total Akt protein levels were quantified by western blot and normalized to GAPDH. n=3 replicates per condition. (H) Quantification of the number of EdU+ MuSCs cultured for 3 d after isolation from uninjured muscle of aged mice with media containing Veh. or WISP1 with or without 0.1 μM of the Akt inhibitor MK-2206. (A, B and H) Cells pooled from up to 3 mice and n≥16 replicates per condition, repeated twice for each condition. (A, B, E, G and H) Data are represented as means ± S.E.M. p-values are *p<0.05, **p<0.01, ***p<0.001 using a Mann-Whitney test when comparing two conditions, and an ANOVA followed by a Bonferroni post hoc test when comparing multiple conditions. See also Figure S7.

Journal: Cell stem cell

Article Title: Aging Disrupts Muscle Stem Cell Function by Impairing Matricellular WISP1 Secretion from Fibro-Adipogenic Progenitors

doi: 10.1016/j.stem.2018.12.014

Figure Lengend Snippet: (A) Quantification of the number of MuSCs cultured for 36 h after isolation from uninjured muscles of aged mice in media containing vehicle (Veh.) or 8 μg/ml WISP1. (B) Quantification of the number of EdU+ MuSCs cultured for 3 days after isolation from uninjured muscles of aged mice in media containing vehicle (Veh.) or WISP1. (C) Representative images of EdU+ MuSCs cultured for 3 days after isolation from uninjured muscles of aged mice in media containing vehicle (Veh.) or WISP1. Scale bars = 50 μm. (D) Pax7 and YFP immunostaining of an asymmetric MuSC division on a single myofiber from a Myf5-Cre/R26R-YFP mouse. Scale bar = 25 μm (top), 5 μm (bottom). (E) Quantification of the number of symmetric (YFP−/YFP−) and asymmetric (YFP−/YFP+) divisions per single myofiber cultured in media containing Veh. or WISP1 for 42 h after isolation from adult uninjured EDL muscles of Myf5-Cre/R26R-YFP mice. n=3 mice per condition, n≥30 fibers analyzed per mouse. (F and G) Primary myoblasts were treated with either Veh. or WISP1 for 24 h and phospho-Akt (p-Akt) and total Akt protein levels were quantified by western blot and normalized to GAPDH. n=3 replicates per condition. (H) Quantification of the number of EdU+ MuSCs cultured for 3 d after isolation from uninjured muscle of aged mice with media containing Veh. or WISP1 with or without 0.1 μM of the Akt inhibitor MK-2206. (A, B and H) Cells pooled from up to 3 mice and n≥16 replicates per condition, repeated twice for each condition. (A, B, E, G and H) Data are represented as means ± S.E.M. p-values are *p<0.05, **p<0.01, ***p<0.001 using a Mann-Whitney test when comparing two conditions, and an ANOVA followed by a Bonferroni post hoc test when comparing multiple conditions. See also Figure S7.

Techniques: Cell Culture, Isolation, Immunostaining, Western Blot, MANN-WHITNEY

(A) Experimental overview of the in-vivo transplantation of FAPs in WISP1−/− recipient mice. (B and C) Quantification and representative images of Pax7+/MyoD+ MuSCs by immunofluorescence in muscle cross-sections of WISP1−/− mice at 4dpi, after intra-muscular injection of vehicle or FAPs freshly isolated from young, aged or WISP1−/− mice at 1 dpi. n≥3 mice per condition. (D) Experimental overview of the in-vivo transplantation of FAPs in aged WT recipient mice. (E) Quantification of the number of Pax7+/MyoD+ MuSCs by immunofluorescence in muscle cross-sections of aged WT mice at 4dpi, after intra-muscular injection of vehicle or FAPs freshly isolated from young, aged or WISP1−/− mice at 1 dpi. n≥4 mice per condition. (B and E) Data are represented as means ± S.E.M. p-values are *<0.05, **p<0.01 using an ANOVA followed by a Bonferroni post hoc test.

Journal: Cell stem cell

Article Title: Aging Disrupts Muscle Stem Cell Function by Impairing Matricellular WISP1 Secretion from Fibro-Adipogenic Progenitors

doi: 10.1016/j.stem.2018.12.014

Figure Lengend Snippet: (A) Experimental overview of the in-vivo transplantation of FAPs in WISP1−/− recipient mice. (B and C) Quantification and representative images of Pax7+/MyoD+ MuSCs by immunofluorescence in muscle cross-sections of WISP1−/− mice at 4dpi, after intra-muscular injection of vehicle or FAPs freshly isolated from young, aged or WISP1−/− mice at 1 dpi. n≥3 mice per condition. (D) Experimental overview of the in-vivo transplantation of FAPs in aged WT recipient mice. (E) Quantification of the number of Pax7+/MyoD+ MuSCs by immunofluorescence in muscle cross-sections of aged WT mice at 4dpi, after intra-muscular injection of vehicle or FAPs freshly isolated from young, aged or WISP1−/− mice at 1 dpi. n≥4 mice per condition. (B and E) Data are represented as means ± S.E.M. p-values are *<0.05, **p<0.01 using an ANOVA followed by a Bonferroni post hoc test.

Techniques: In Vivo, Transplantation Assay, Immunofluorescence, Injection, Isolation

(A-C) qPCR quantification of Pax7, MyoD and MyoG mRNA in muscles of aged mice treated with daily i.p. injection of Veh. or WISP1 at 1mg/kg under uninjured (uninj.) conditions or at 3, 7 and 14 dpi. (D) Representative immunostainings for Pax7 and Ki67 of cross sections of Veh. or WISP1 treated aged muscles at 3 dpi. Yellow and blue arrowheads show Pax7+/Ki67+ and Pax7+/Ki67− MuSCs, respectively. Scale bars = 50 μm. (E) Quantification of the number of Pax7+/Ki67+ and Pax7+/Ki67− MuSCs in cross sections of young and Veh. or WISP1 treated aged muscles at 3 dpi. (F) Quantification of the number of Pax7+/MyoD+ MuSCs in cross sections of young and Veh. or WISP1 treated aged muscles at 3 dpi. (G) Representative Laminin and embryonic myosin heavy chain (eMHC) immunostainings of cross sections of Veh. or WISP1 treated aged muscles at 7 dpi. Scale bars = 100 μm. (H) Quantification of the area covered by eMHC positive fibers in sections of young and Veh. or WISP1 treated aged muscles at 7 and 14 dpi. (I) Representative Laminin immunostainings of cross sections of Veh. or WISP1 treated aged muscles at 7 dpi. Scale bars = 100 μm. (J) Quantification of the cross-sectional area distribution of regenerating fibers with centralized nuclei in sections of young and aged Veh. or WISP1 treated muscles at 7 dpi. Inter-class statistics are compared to the aged Veh. group. (A-C, E, F, H and J) n≥4 mice per condition. Data are represented as means ± S.E.M. p-values are *<0.05, **p<0.01, ***p<0.001 using an ANOVA followed by a Bonferroni post hoc test when comparing multiple conditions, and a Kolmogorov-Smirnov test to assess fiber cross-sectional area distributions. See also Figure S8.

Journal: Cell stem cell

Article Title: Aging Disrupts Muscle Stem Cell Function by Impairing Matricellular WISP1 Secretion from Fibro-Adipogenic Progenitors

doi: 10.1016/j.stem.2018.12.014

Figure Lengend Snippet: (A-C) qPCR quantification of Pax7, MyoD and MyoG mRNA in muscles of aged mice treated with daily i.p. injection of Veh. or WISP1 at 1mg/kg under uninjured (uninj.) conditions or at 3, 7 and 14 dpi. (D) Representative immunostainings for Pax7 and Ki67 of cross sections of Veh. or WISP1 treated aged muscles at 3 dpi. Yellow and blue arrowheads show Pax7+/Ki67+ and Pax7+/Ki67− MuSCs, respectively. Scale bars = 50 μm. (E) Quantification of the number of Pax7+/Ki67+ and Pax7+/Ki67− MuSCs in cross sections of young and Veh. or WISP1 treated aged muscles at 3 dpi. (F) Quantification of the number of Pax7+/MyoD+ MuSCs in cross sections of young and Veh. or WISP1 treated aged muscles at 3 dpi. (G) Representative Laminin and embryonic myosin heavy chain (eMHC) immunostainings of cross sections of Veh. or WISP1 treated aged muscles at 7 dpi. Scale bars = 100 μm. (H) Quantification of the area covered by eMHC positive fibers in sections of young and Veh. or WISP1 treated aged muscles at 7 and 14 dpi. (I) Representative Laminin immunostainings of cross sections of Veh. or WISP1 treated aged muscles at 7 dpi. Scale bars = 100 μm. (J) Quantification of the cross-sectional area distribution of regenerating fibers with centralized nuclei in sections of young and aged Veh. or WISP1 treated muscles at 7 dpi. Inter-class statistics are compared to the aged Veh. group. (A-C, E, F, H and J) n≥4 mice per condition. Data are represented as means ± S.E.M. p-values are *<0.05, **p<0.01, ***p<0.001 using an ANOVA followed by a Bonferroni post hoc test when comparing multiple conditions, and a Kolmogorov-Smirnov test to assess fiber cross-sectional area distributions. See also Figure S8.

Techniques: Injection

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1) Product Images from "Matricellular Protein WISP2 Is an Endogenous Inhibitor of Collagen Linearization and Cancer Metastasis"

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