Journal: bioRxiv

Article Title: WISP1 drives a mechanically active immune modulatory and proliferative cardiac myofibroblast state

doi: 10.64898/2026.02.17.706476

Figure Lengend Snippet: (A) Wisp1 mRNA expression in primary adult mouse cardiac fibroblasts following TGFβ1 (10 ng/mL) treatment for 24–72 h. (B) Representative immunofluorescence images of α-SMA (green), vimentin (red), and DAPI (blue) after 72 h treatment with vehicle (Ctrl), WISP1 (500 ng/mL), TGFβ1 (10 ng/mL), or WISP1 + TGFβ1. (C, D) Representative Western blot (C) and densitometric quantification (D) of α-SMA normalized to GAPDH. Data are mean ± SEM from ≥3 independent isolations. Statistical analysis by one-way ANOVA with Tukey’s post hoc test; *p < 0.05. Data are mean ± SEM from ≥3 independent isolations. Statistical analysis by one-way ANOVA with Tukey’s post hoc test; *p < 0.05.

Article Snippet: For treatments, CFs were seeded in 12- or 24-well plates in DMEM + 10% FBS and treated for 72 h with recombinant human TGFβ1 (10 ng/mL; R&D Systems, 7754-BH) and/or recombinant murine WISP1 (500 ng/mL; R&D Systems, 1680-WS-050).

Techniques: Expressing, Immunofluorescence, Western Blot

Journal: bioRxiv

Article Title: WISP1 drives a mechanically active immune modulatory and proliferative cardiac myofibroblast state

doi: 10.64898/2026.02.17.706476

Figure Lengend Snippet: Primary adult mouse cardiac fibroblasts were embedded in floating collagen gels and treated for 24 h with vehicle (Ctrl), WISP1 (500 ng/mL), TGFβ1 (10 ng/mL), or WISP1 + TGFβ1. (A) Representative gel images at 0 h and 24 h. (B) Quantification of gel area (% contraction; n = 4 isolations, 2 males & 2 females). (C–D) Confluent fibroblast monolayers were scratched and imaged every 2 hours for 24 hours using the same treatment conditions. (C) Representative bright-field images at time point 0 and 14. (D) Quantification of wound closure (% of gap closed relative to time 0; n = 8 isolations, 4 male & 4 female). Data are mean ± SEM. One-way ANOVA with Tukey’s multiple comparisons; *p ≤ 0.05.

Article Snippet: For treatments, CFs were seeded in 12- or 24-well plates in DMEM + 10% FBS and treated for 72 h with recombinant human TGFβ1 (10 ng/mL; R&D Systems, 7754-BH) and/or recombinant murine WISP1 (500 ng/mL; R&D Systems, 1680-WS-050).

Techniques:

Journal: iScience

Article Title: WNT1-inducible signaling pathway protein 1 activation through C-X-C motif chemokine ligand 5/C-X-C chemokine receptor type 2/leukemia inhibitory factor/leukemia inhibitory factor receptor signaling promotes immunosuppression and neuroendocrine differentiation in prostate cancer.

doi: 10.1016/j.isci.2024.110562

Figure Lengend Snippet: Figure 3. Knockdown of WPMY-1 suppresses LIF-driven neuroendocrine differentiation (NED) and malignant progression in prostate cancer (PCa) cells (A) Relative protein levels of LIF, LIFR, WISP1, SOCS3, and PDL1 were measured in AR-positive PCa cell lines (LNCaP, C4-2, and 22Rv1), an AR-negative PCa cell line (PC3), and an NEPC cell line (LASCPC-01). (B) Relative protein levels of WISP1, phosphorylated (p)-STAT3, STAT3, PDL1, and SOCS3 were measured in LNCaP cells treated with PBS or 100 ng/mL of the LIF recombinant protein for a duration ranging 0 to 96 h. (C) Relative mRNA levels of WISP1, neuroendocrine (CHGA, SYP, and ENO2), stem cell (SOX2 and NANOG), and anti-inflammatory (SOCS3 and PDL1) markers in LNCaP cells treated with PBS or 100 ng/mL of the LIF recombinant protein or combined treatment with DMSO or 35 nM EC330 for 48 h * vs. PBS+DMSO; # vs. LIF+DMSO, as determined by a one-way ANOVA. (D) Relative protein levels of WISP1, p-STAT3, STAT3, PDL1, and SOCS3 in LNCaP and C4-2 cells cultured in PBS or 100 ng/mL of the LIF recombinant protein, followed by DMSO or 35 nM EC330 treatment for 48 h. (E) Relative mRNA expression levels of WISP1, neuroendocrine markers, stem cell markers, and anti-inflammatory markers (SOCS3 and PDL1) were measured in LNCaP cells expressing the non-target control (NC) or WISP1 siRNA, followed by treatment with PBS or 100 ng/mL of the LIF recombinant protein for 48 h * vs. PBS+NC; # vs. LIF+NC, as determined by a one-way ANOVA. (F and G) Relative cell proliferation (F) and sphere formation (G) were measured in LNCaP cells expressing either NC or WISP1 siRNA, followed by treatment with either PBS or 100 ng/mL of LIF recombinant protein for 5 days (F) or 1 week (G). Scale bars represent 100 mm (G). * vs. PBS+NC; # vs. LIF+NC, as determined by a one-way ANOVA. (H and I) Relative cell migration (H) and invasion through Matrigel (I) were measured in LNCaP cells expressing the NC or WISP1 siRNA, followed by treatment with PBS or 100 ng/mL of LIF recombinant protein for 12 h. Scale bars representing 20 mm are shown. * vs. PBS+NC; # vs. LIF+NC, as determined by a one-way ANOVA. Quantification of relative mRNA levels, cell proliferation, sphere formation, and cell invasion through Matrigel is presented as the mean G SEM from three biological replicates. *p < 0.05, **p < 0.01, ***p < 0.005. (J) A GSEA of the TCGA PCa dataset revealed significant associations between high WISP1 expression in prostate tissues and a gene signature representing NEPC-responsive signaling. NES, normalized enrichment score; FDR, false discovery rate.

Article Snippet: Protein levels of WISP1 were quantified using a human WISP1 ELISA kit (Elabscience, #E-EL-H5542) following the manufacturer’s instructions.

Techniques: Knockdown, Recombinant, Cell Culture, Expressing, Control, Migration

Journal: iScience

Article Title: WNT1-inducible signaling pathway protein 1 activation through C-X-C motif chemokine ligand 5/C-X-C chemokine receptor type 2/leukemia inhibitory factor/leukemia inhibitory factor receptor signaling promotes immunosuppression and neuroendocrine differentiation in prostate cancer.

doi: 10.1016/j.isci.2024.110562

Figure Lengend Snippet: Figure 4. Crosstalk between prostate cancer (PCa) cells and prostate stromal cells promotes neuroendocrine differentiation (NED) and expressions of immunosuppressive cytokines in the tumor microenvironment (TME) through the activation of LIF/LIFR signaling (A) Relative mRNA expression levels of WISP1, neuroendocrine markers (CHGA, SYP, and ENO2), stem cell markers (SOX2 and NANOG), anti-inflammatory markers (SOCS3 and PDL1), LIF, CXCR2, and CXCL5 in LNCaP cells. These cells were cultured with conditioned medium (CM) collected from human WPMY-1 stromal cells, at concentrations of 0%, 15%, or 50%, for a duration of 48 h * vs. 0%, as determined by a one-way ANOVA. (B) Relative mRNA expression levels of WISP1, neuroendocrine markers, stem cell markers, anti-inflammatory markers, LIF, CXCR2, and CXCL5 were measured in LNCaP cells. These cells were cultured with CM obtained from WPMY-1 stromal cells treated with DMSO or 35 nM EC330 for 48 h * vs. Veh+DMSO; # vs. WPMY-1 CM + DMSO, as determined by a one-way ANOVA. (C) Relative mRNA expression levels of WISP1, neuroendocrine, stem cell, anti-inflammatory markers, LIF, CXCR2, and CXCL5 in LNCaP cells cultured with CM collected from the non-target control (Luc) or LIFR shRNA-expressing WPMY-1 cells for 48 h * vs. Veh; # vs. WPMY-1/shLuc CM, as determined by a one-way ANOVA. (D) Relative mRNA expression levels of LIFR, LIF, CXCL5, WISP1, and anti-inflammatory markers (IL10, IL4, IL1RN, TGFB1, VEGFA, IFNA17, and SOCS3) in WPMY-1 cells stably expressing the shLuc or LIFR shRNA. * vs. shLuc, as determined by a one-way ANOVA. (E) Relative mRNA expression levels of LIFR, LIF, CXCL5, WISP1, and anti-inflammatory markers in WPMY-1 cells treated with PBS or 100 ng/mL of the LIF recombinant protein, followed by DMSO or 35 nM EC330 treatment for 48 h * vs. PBS+DMSO; # vs. LIF+DMSO, as determined by a one-way ANOVA. (F and G) Cell proliferation (F) and sphere formation (G) of LNCaP cells were evaluated. These cells were cultured with CM obtained from WPMY-1 stromal cells treated with DMOS or 35 nM EC330 for 48 h. Scale bars in (G) represent 100 mm. Statistical comparisons were performed using a one-way ANOVA. * vs. Veh+DMSO; # vs. WPMY-1 CM + DMSO. (H and I) Relative cell migration (H) and invasion through Matrigel (I) were measured in LNCaP cells. These cells were cultured with CM obtained from WPMY-1 stromal cells treated with DMSO or 35 nM EC330 for 12 h. Scale bars representing 20 mm are shown. * vs. Veh+DMSO; # vs. WPMY-1 CM + DMSO, as determined by a one-way ANOVA. Quantification of relative mRNA levels, cell proliferation, sphere formation, and cell invasion through Matrigel is presented as the mean G SEM, based on three biological replicates. Significance levels are denoted as *p < 0.05, **p < 0.01, and ***p < 0.005.

Article Snippet: Protein levels of WISP1 were quantified using a human WISP1 ELISA kit (Elabscience, #E-EL-H5542) following the manufacturer’s instructions.

Techniques: Activation Assay, Expressing, Cell Culture, Control, shRNA, Stable Transfection, Recombinant, Migration

Journal: iScience

Article Title: WNT1-inducible signaling pathway protein 1 activation through C-X-C motif chemokine ligand 5/C-X-C chemokine receptor type 2/leukemia inhibitory factor/leukemia inhibitory factor receptor signaling promotes immunosuppression and neuroendocrine differentiation in prostate cancer.

doi: 10.1016/j.isci.2024.110562

Figure Lengend Snippet: Figure 5. LIF/STAT3-driven transcription of WISP1 through direct binding to the gamma interferon activation site (GAS) of the regulatory sequence (A) ChIP-sequencing analysis was performed to detect the GAS for WISP1. Detected GAS sites are labeled as black boxes in the tracks. ChIP-sequencing data were downloaded from Gene Expression Omnibus (GEO) (GSM2752900) and analyzed using the Genome Browser (Genomics Institute, UCSC). (B) A schematic representation is shown for the predicted wild-type (WT) and mutant (M)-GASs in the regulatory sequence reporter constructs of the human WISP1 gene (GRCh38:8) (C and D) A ChIP assay was performed to show the binding of phosphorylated (p)-STAT3 to the predicted GAS in the WISP1 gene regulatory sequence. The assay was conducted in LNCaP cells (C) or WPMY-1 cells (D) treated with PBS or 100 ng/mL of the LIF recombinant protein, followed by treatment with DMSO or with 10 or 35 nM EC330 for 48 h. Sheared chromatin from nuclear extracts was precipitated with antibodies to p-STAT3 or control IgG, and predictive primers (B, indicated by black arrows) were used to quantify the precipitated DNA using a qPCR. Enrichment of each protein at each site is presented as a percentage of the total input and then normalized to IgG. * vs. PBS+DMSO; # vs. LIF+DMSO, as determined by a one-way ANOVA. (E) A ChIP assay was performed to demonstrate the binding of p-STAT3 to the predicted GAS in the regulatory sequence of the LIF gene in WPMY-1 cells. Cells were treated with PBS or 100 ng/mL of the LIF recombinant protein, followed by treatment with DMSO or with 10 or 35 nM EC330 for 48 h * vs. PBS+DMSO; # vs. LIF+DMSO, as determined by a one-way ANOVA. (F–I) The relative mean fluorescence intensity (MFI) of the GFP reporter gene containing either the WT-GAS or M-GAS from the WISP1 regulatory sequence was measured in LNCaP cells (F and G) and WPMY-1 cells (H and I) after treatment with PBS or 100 ng/mL of LIF recombinant protein. This was followed by further treatment with either DMSO or EC330 (10 or 35 nM) for 48 h (F and H). * vs. WT/PBS+DMSO (F and H) or WT + PBS (G and I); # vs. WT/LIF+DMSO (F and H) or WT + LIF (G and I), as determined by a one-way ANOVA. (J and K) The relative MFI of the GFP reporter gene containing the WT-GAS or M-GAS from the LIF regulatory sequence was measured in WPMY-1 cells treated with PBS or 100 ng/mL of LIF recombinant protein or combined treatment with DMSO or 10 or 35 nM EC330 for 48 h * vs. WT/PBS+DMSO (J) or WT + PBS (K); # vs. WT/LIF+DMSO (J) or WT + LIF (K), as determined by a one-way ANOVA. Quantification of relative p-STAT3 enrichment and MFI is presented as the mean G SEM from three biological replicates. *p < 0.05, **p < 0.01, ***p < 0.005.

Article Snippet: Protein levels of WISP1 were quantified using a human WISP1 ELISA kit (Elabscience, #E-EL-H5542) following the manufacturer’s instructions.

Techniques: Binding Assay, Activation Assay, Sequencing, ChIP-sequencing, Labeling, Gene Expression, Mutagenesis, Construct, Recombinant, Control

Journal: iScience

Article Title: WNT1-inducible signaling pathway protein 1 activation through C-X-C motif chemokine ligand 5/C-X-C chemokine receptor type 2/leukemia inhibitory factor/leukemia inhibitory factor receptor signaling promotes immunosuppression and neuroendocrine differentiation in prostate cancer.

doi: 10.1016/j.isci.2024.110562

Figure Lengend Snippet: Figure 6. WISP1 abundance in serum relative to prostate cancer (PCa) progression (A) Relative mRNA levels of CXCL5, CXCR2, LIF, WISP1, neuroendocrine (CHGA, SYP, and ENO2), and stem cell (SOX2 and NANOG) markers in C4-2 cells expressing the empty vector (EV) or CXCL5-expressing vector, followed by treatment with DMSO or 35 nM EC330 for 48 h * vs. EV + DMSO; # vs. CXCL5+DMSO, as determined by a one-way ANOVA. (B and C) Tumor growth analysis was conducted by subcutaneously inoculating male nude mice with C4-2 cells expressing either the EV or a CXCL5-expressing vector. The mice were then treated bi-daily with either DMSO or 2.5 mg/kg EC330 via intraperitoneal injection and allowed to grow for 8 weeks. Tumor sizes were measured weekly (B). Tumor weights were measured upon tumor collection (C). n = 5 per group. * vs. EV + DMSO; # vs. CXCL5+DMSO, as determined by a one- way ANOVA and t-test. (D and E) Immunohistochemical (IHC) staining and intensity analyses were performed to assess protein levels of CXCL5, CXCR2, LIF, WISP1, and ENO2 in subcutaneous tumors derived from (B). * vs. EV + DMSO; # vs. CXCL5+DMSO, as determined by a two-tailed Student’s t-test. Scale bars, 100 mm. (F) WISP1 concentrations were measured in patient sera derived from samples of benign prostatic hyperplasia (BPH; n = 10), hormone-sensitive PCa (HSPC, n = 10), and metastatic castration-resistant PCa (mCRPC; n = 8). * vs. BPH; # vs. HSPC, analyzed by a one-way ANOVA.

Article Snippet: Protein levels of WISP1 were quantified using a human WISP1 ELISA kit (Elabscience, #E-EL-H5542) following the manufacturer’s instructions.

Techniques: Expressing, Plasmid Preparation, Injection, Immunohistochemical staining, Immunohistochemistry, Derivative Assay, Two Tailed Test

Journal: Clinical Science (London, England : 1979)

Article Title: WNT1-inducible signaling pathway protein 1 regulates kidney inflammation through the NF-κB pathway

doi: 10.1042/CS20210663

Figure Lengend Snippet: ( A ) F4/80 positive cells were accumulated in mice kidney (kidney cortex) with WISP1 administration and reduced by PTL, which was verified by counting the percentage of F4/80 positive cells. ( B ) The expression of inflammation markers was up-regulated by WISP1 and alleviated by PTL treatment. ( C ) M1 and M2 marker expression. Results are expressed as the mean ± SEM relative gene expression, *** P <0.001,** P <0.01, * P <0.05. n =3–5.

Article Snippet: Recombinant human WISP1 protein, IgG control and mouse WISP1 antibody were purchased commercially (R&D Systems, Minneapolis, MN) and used at specified concentrations.

Techniques: Expressing, Marker, Gene Expression

Journal: Clinical Science (London, England : 1979)

Article Title: WNT1-inducible signaling pathway protein 1 regulates kidney inflammation through the NF-κB pathway

doi: 10.1042/CS20210663

Figure Lengend Snippet: At 48 h after administration of WISP1 and/or PTL to healthy mice, there were: ( A ) no morphology changes (Masson staining) observed based on in the kidney at 48 h after administration of WISP1 and/or PTL in healthy mice. ( B ) No collagen (PSR staining) alteration was found in the WISP1-treated mice. ( C ) Q-PCR data showed no significant changes in collagen1a1 and fibronectin. ( D ) α-SMA expression were up-regulated with WISP1 treatment, reduced by PTL. Results are expressed as the mean ± SEM relative gene expression, ** P <0.01, * P <0.05. n =3–5.

Article Snippet: Recombinant human WISP1 protein, IgG control and mouse WISP1 antibody were purchased commercially (R&D Systems, Minneapolis, MN) and used at specified concentrations.

Techniques: Staining, Expressing, Gene Expression

Journal: Clinical Science (London, England : 1979)

Article Title: WNT1-inducible signaling pathway protein 1 regulates kidney inflammation through the NF-κB pathway

doi: 10.1042/CS20210663

Figure Lengend Snippet: ( A ) The translocation of P-p65 into nucleus induced by WISP1 was inhibited by treatment of NFκB inhibitor PTL, with the inhibition on the up-regulated expression of TNF-α. ( B , C ) PTL inhibited the LPS-induced nucleus translocation of P-p65 and the up-regulated expression of inflammation markers. ( D ) Results are expressed as the mean ± SEM relative gene expression, *** P <0.001,** P <0.01. n =3–5.

Article Snippet: Recombinant human WISP1 protein, IgG control and mouse WISP1 antibody were purchased commercially (R&D Systems, Minneapolis, MN) and used at specified concentrations.

Techniques: Translocation Assay, Inhibition, Expressing, Gene Expression

Journal: Clinical Science (London, England : 1979)

Article Title: WNT1-inducible signaling pathway protein 1 regulates kidney inflammation through the NF-κB pathway

doi: 10.1042/CS20210663

Figure Lengend Snippet: ( A ) Knockdown (KD) of WISP1 by shRNA. ( B ) Knockdown of WISP1 in LPS-stimulated RAW macrophages inhibited ( B ) the translocation of P-p65 into nucleus, and up-regulated ( C ) expression of inflammation markers. *** P <0.001,** P <0.01. n =3–5.

Article Snippet: Recombinant human WISP1 protein, IgG control and mouse WISP1 antibody were purchased commercially (R&D Systems, Minneapolis, MN) and used at specified concentrations.

Techniques: Knockdown, shRNA, Translocation Assay, Expressing

Journal: Clinical Science (London, England : 1979)

Article Title: WNT1-inducible signaling pathway protein 1 regulates kidney inflammation through the NF-κB pathway

doi: 10.1042/CS20210663

Figure Lengend Snippet: ( A ) The addition of WISP1 antibody (Ab) inhibited the translocation of P-p65 induced by LPS and the expression of inflammation markers ( B,C ). ELISA showed that the up-regulation TNF-α was reduced by WISP1 knockdown and the addition of antibody. Results are expressed as the mean ± SEM relative gene expression, *** P <0.001,** P <0.01, * P <0.05. n =3–5.

Article Snippet: Recombinant human WISP1 protein, IgG control and mouse WISP1 antibody were purchased commercially (R&D Systems, Minneapolis, MN) and used at specified concentrations.

Techniques: Translocation Assay, Expressing, Enzyme-linked Immunosorbent Assay, Knockdown, Gene Expression

Journal: Clinical Science (London, England : 1979)

Article Title: WNT1-inducible signaling pathway protein 1 regulates kidney inflammation through the NF-κB pathway

doi: 10.1042/CS20210663

Figure Lengend Snippet: The representative cell images were taken using 4× light microscope. ( A ) PTL inhibited kidney fibroblast cells proliferation induced by recombinant WISP1 protein (1 μg/ml), validated by counting cell numbers. ( B ) Wisp1 knocked-down using shRNA reduced the proliferation of kidney fibroblast cells by TNF-α, confirmed by counting cell number. ( C ) The addition of WISP1 antibody reduced the fibroblast cell growth in the presence of TNF-α, as confirmed by counting cell number. n =3.

Article Snippet: Recombinant human WISP1 protein, IgG control and mouse WISP1 antibody were purchased commercially (R&D Systems, Minneapolis, MN) and used at specified concentrations.

Techniques: Light Microscopy, Recombinant, shRNA

Journal: Clinical Science (London, England : 1979)

Article Title: WNT1-inducible signaling pathway protein 1 regulates kidney inflammation through the NF-κB pathway

doi: 10.1042/CS20210663

Figure Lengend Snippet: ( A ) STZ-induced DN mice at 18 weeks demonstrated the increased expression of TNF-α, MCP-1, CD68, α-SMA and WISP1 gene by qPCR, compared with control mice ( n =12–15 mice/group). Immunofluorescence staining indicated the elevated expression of WISP1 in DN model. Macrophage co-localized with WISP1 staining (white arrow). ( B ) High glucose-treated RAW cells expressed more WISP1, TNF-α and MCP-1 than low glucose-treated cells. ( C ) The kidneys of mice with 7 days post-UUO surgery showed an increased expression of inflammation genes, TNF-α, MCP-1 and IL6 and an up-regulation of WISP1, compared with control kidneys ( n =5 mice/group). Immunofluorescence staining indicated the elevated expression of WISP1 in UUO model. Macrophage co-localized with WISP1 staining (white arrow). *** P <0.001,** P <0.01.

Article Snippet: Recombinant human WISP1 protein, IgG control and mouse WISP1 antibody were purchased commercially (R&D Systems, Minneapolis, MN) and used at specified concentrations.

Techniques: Expressing, Control, Immunofluorescence, Staining

Journal: Clinical Science (London, England : 1979)

Article Title: WNT1-inducible signaling pathway protein 1 regulates kidney inflammation through the NF-κB pathway

doi: 10.1042/CS20210663

Figure Lengend Snippet: ( A ) Q-PCR analysis demonstrated that WISP1 antibody reduced inflammation gene expression in kidneys from UUO mice. ( B ) UUO kidneys without treatment or receiving IgG show an up-regulated F4/80 positive cells accumulation at 7 days. Administration of WISP1 antibody in UUO mice resulted in a reduction, verified by the percentage of F4/80 positive cells. ( C ) WISP1 antibody treatment rescued the degradation of IκBα expression in UUO kidneys, by Western blotting. Results are expressed as the mean ± SEM relative gene expression, *** P <0.001, ** P <0.01, * P <0.05. n =3–7.

Article Snippet: Recombinant human WISP1 protein, IgG control and mouse WISP1 antibody were purchased commercially (R&D Systems, Minneapolis, MN) and used at specified concentrations.

Techniques: Gene Expression, Expressing, Western Blot

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, Clinical Proteomics, Expressing, Gene Expression, Two Tailed Test

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

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

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

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, Control, Expressing, Infection

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, Phospho-proteomics, Western Blot, Incubation, Recombinant, Molecular Weight, Control, Immunofluorescence, Staining, Isolation, Two Tailed Test

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, Clinical Proteomics, Expressing, Gene Expression, Two Tailed Test

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

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

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

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, Control, Expressing, Infection

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, Phospho-proteomics, Western Blot, Incubation, Recombinant, Molecular Weight, Control, Immunofluorescence, Staining, Isolation, Two Tailed Test