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: Mouse Wisp1 levels were determined using a Mouse/Rat Wisp-1/CCN4 Immunoassay (R&D Systems) that detects both natural and full-length recombinant Wisp1 protein.

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: Mouse Wisp1 levels were determined using a Mouse/Rat Wisp-1/CCN4 Immunoassay (R&D Systems) that detects both natural and full-length recombinant Wisp1 protein.

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: Mouse Wisp1 levels were determined using a Mouse/Rat Wisp-1/CCN4 Immunoassay (R&D Systems) that detects both natural and full-length recombinant Wisp1 protein.

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: Mouse Wisp1 levels were determined using a Mouse/Rat Wisp-1/CCN4 Immunoassay (R&D Systems) that detects both natural and full-length recombinant Wisp1 protein.

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: Mouse Wisp1 levels were determined using a Mouse/Rat Wisp-1/CCN4 Immunoassay (R&D Systems) that detects both natural and full-length recombinant Wisp1 protein.

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: Mouse Wisp1 levels were determined using a Mouse/Rat Wisp-1/CCN4 Immunoassay (R&D Systems) that detects both natural and full-length recombinant Wisp1 protein.

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

Journal: Oncotarget

Article Title: Modulation of macrophage polarization and lung cancer cell stemness by MUC1 and development of a related small-molecule inhibitor pterostilbene

doi: 10.18632/oncotarget.8101

Figure Lengend Snippet: A. Using Oncomine database search, we found a significant increased level of Muc-1 (8.478-fold increase) was detected in lung adenocarcinoma ( N = 39) as compared to normal lung tissues such as fetal lung ( N = 1) and adult lung samples ( N = 5). Garber lung Database was selected. B. PrognoScan analysis indicated that lung cancer patients with high Muc-1 expression ( n = 63) were associated with a poor overall survival rate as compared to patients with lower or no Muc-1 expression ( n = 75). C. Representative immunohistochemistry photomicrographs of MUC1 (cytoplasmic) or macrophage marker CD68 expression in NSCLC. Arrow indicates CD68 + TAM.

Article Snippet: The MUC1 expression level was assayed using MUC1 (VU4H5) Mouse mAb #4538 (Cell Signaling, Danvers, MA).

Techniques: Expressing, Immunohistochemistry, Marker

Journal: Oncotarget

Article Title: Modulation of macrophage polarization and lung cancer cell stemness by MUC1 and development of a related small-molecule inhibitor pterostilbene

doi: 10.18632/oncotarget.8101

Figure Lengend Snippet: Expression of MUC1 and CD68 in lung adenocarcinoma specimens

Article Snippet: The MUC1 expression level was assayed using MUC1 (VU4H5) Mouse mAb #4538 (Cell Signaling, Danvers, MA).

Techniques: Expressing

Journal: Oncotarget

Article Title: Modulation of macrophage polarization and lung cancer cell stemness by MUC1 and development of a related small-molecule inhibitor pterostilbene

doi: 10.18632/oncotarget.8101

Figure Lengend Snippet: A. FACS analysis demonstrates that the CD133 + cell population increased in both A549 (from 3.86 to 8.07%) and H441 (from 5.68 to 10.80%) in the presence of M2 TAM. B. Gene profile was also changed in the presence of M2 TAMs. MUC1, Sox2 (stemness), NF-κB (inflammation) expression level were increased in the presence of M2 TAMs while a major EMT marker, E-cadherin was downregulated. C. Sphere forming assay of sphere-forming assay of A549 and H441 cells in the presence of M2 TAM. Cells (1×10 3 cells/ml) were cultured in serum-free DMEM/F12 medium with growth factors (10 ng/ml of EGF and bFGF each) for 10 days and counted the spheres as described in ‘Materials and methods’. Representative images of spheres (left magnification, 100x) and quantification of the assay (right).

Article Snippet: The MUC1 expression level was assayed using MUC1 (VU4H5) Mouse mAb #4538 (Cell Signaling, Danvers, MA).

Techniques: Expressing, Marker, Cell Culture

Journal: Oncotarget

Article Title: Modulation of macrophage polarization and lung cancer cell stemness by MUC1 and development of a related small-molecule inhibitor pterostilbene

doi: 10.18632/oncotarget.8101

Figure Lengend Snippet: A. Pterostilbene treatment suppressed the Muc1 and NF-κB expression in M2 polarization of THP-1 cells. B . Muc1, NF-κB and VEGF mRNA expression levels in M2 polarization of THP-1 cells were significantly decreased after co-culturing with pterostilbene in H441 cells. C . FACS analysis indicated that in the presence of pterostilbene, the percentage of ALDH subpopulation cells in TAM co-cultured H441 was significantly decreased.

Article Snippet: The MUC1 expression level was assayed using MUC1 (VU4H5) Mouse mAb #4538 (Cell Signaling, Danvers, MA).

Techniques: Expressing, Cell Culture

Journal: Oncotarget

Article Title: Modulation of macrophage polarization and lung cancer cell stemness by MUC1 and development of a related small-molecule inhibitor pterostilbene

doi: 10.18632/oncotarget.8101

Figure Lengend Snippet: A. A549 and H441 cells were co-incubated with wild-type (WT) M2 TAMs and Muc1-silenced (KD) TAMs and analyzed for their stemness. FACS results demonstrated that the percentage of CD133 + cells did significant increase as in ones co-incubated with the wild-type M2 TAMs. B. Western blots of A549+M2 and H441+M2 cells treated with Muc1 siRNA demonstrated that the expression levels of p65 (NF-κB) and vimentin were downregulated as compared to that in the cells that received control siRNA. Comparative gene profiling of NSCLC cells co-incubated with wildtype (WT) and Muc1 knock-down (KD) TAMs. Data indicated that Muc1-silenced TAMs did not induced stemness in A549 and H441 cells. CD133, Sox2, NF-κB expression level was comparatively lower in the Muc1 KD groups as compared to those in WT counterparts. Gene expression analysis of selected M1 and M2-associated transcripts in macrophages after being treated with control siRNA or MUC1 siRNA in THP-1 cells. C. Self-renewal ability was compromised in Muc1 KD M2 co-cultured NSCLCs. Both A549 and H441 showed decreased tumor sphere forming ability when co-incubated with Muc1 KD TAMs. D. Proposed mechanistic actions of Muc1 in M2 polarization and promoting stemness in NSCLCs.

Article Snippet: The MUC1 expression level was assayed using MUC1 (VU4H5) Mouse mAb #4538 (Cell Signaling, Danvers, MA).

Techniques: Incubation, Western Blot, Expressing, Control, Knockdown, Gene Expression, Cell Culture

Journal: BMC Biology

Article Title: Reengineering anthrax toxin protective antigen for improved receptor-specific protein delivery

doi: 10.1186/s12915-020-00827-y

Figure Lengend Snippet: Effects of PA on different cell lines expressing EpCAM. a EpCAM surface expression data assessed via flow cytometry using an Alexa Fluor 488-labeled anti-EpCAM mouse mAb ( n = 3). Error bars reflect SEM. b Confocal imaging of stained Flp-In 293-EpCAM-BirA cells with PA wt -sANTXR-Ac2 and LF N -eGFP to assess PA oligomerization. c Viability assays for a set of cell lines with PA wt -sANTXR-Ac2, PA m -sANTXR-Ac2, PA m -Ac2, PA wt -Ac2, and PA wt ( n = 3). Error bars reflect SEM

Article Snippet: Cells were stained with Alexa Fluor 488-labeled anti-EpCAM mouse mAb (VU1D9, Cell Signaling) for EpCAM levels.

Techniques: Expressing, Flow Cytometry, Labeling, Imaging, Staining