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: Aging (Albany NY)

Article Title: HNRNPA1-mediated 3′ UTR length changes of HN1 contributes to cancer- and senescence-associated phenotypes

doi: 10.18632/aging.102060

Figure Lengend Snippet: Knockdown of HN1 induces cellular senescence in normal and cancer cells. ( A – B ) Validation of HN1 knockdown (KD) in HUVEC with two shRNAs (shHN1_#1, shHN1_#2), as quantified by qRT-PCR ( A ) and Western blot ( B ), respectively. GAPDH served as internal control for both mRNA and protein. ( C ) Cell proliferation rate evaluated by Cell Counting Kit-8 (CCK-8) assay in HN1 -KD HUVEC cells with two shRNAs. *, ** and *** stand for p < 0.05, p < 0.01 and p < 0.001, respectively, based on t -test with three biological replicates. ( D – E ) Representative SA-β-Gal staining (D) and quantitative statistics (E) in HN1 -KD and control HUVEC cells. *** represents p < 0.001 based on t -test with three independent countings. ( F – G ) EdU incorporation assay ( F ) and quantitative statistics ( G ) in HN1 -KD and control HUVEC cells. Green and blue dots stand for incorporated EdU and DNA DAPI (4',6-diamidino-2-phenylindole) staining, respectively. * and *** stand for p < 0.05 and p < 0.001, respectively, based on t -test with three independent countings. ( H – I ) Validation of HN1 knockdown in A549 cells, as described in panel A-B. ( J ) Cell proliferation rate evaluated by CCK-8 assay in HN1 -KD A549 cells, as described in panel C. ( K – L ) SA-β-Gal staining ( K ) and quantitative statistics ( L ) were shown in A549 cells, as described in panel D-E. ** represents p < 0.01 based on t -test with three independent countings. ( M – N ) EdU incorporation assay ( M ) and positive cell (EdU incorporated) statistics ( N ) were shown in A549 cells, as described in panel F-G. ** represents p < 0.01 based on t -test with three independent countings. ( O – Q ) Over-expression of HN1 partially rescued HN1 -KD induced SA-β-Gal activity in HUVECs. qRT-PCR confirmed overexpression of HN1 in HN1 -KD HUVECs ( O ). *** represents p < 0.001 based on t -test with three qPCR reactions. SA-β-Gal staining ( P ), and positive staining cell statistics ( Q ) in control (+MOCK) and overexpression (+ HN1 ) HUVECs. *** represents p < 0.001 based on t -test with three independent countings. ( R – T ) Over-expression of HN1 partially rescued HN1 -KD induced SA-β-Gal activity in A549 cells. qRT-PCR confirmed overexpression of HN1 in HN1 -KD A549 cells ( R ). SA-β-Gal staining ( S ) and positive staining cell statistics ( T ) in control (+MOCK) and overexpression (+ HN1 ) A549 cells. *** represents p < 0.001, as described in panel O-Q.

Article Snippet: Membranes were incubated with anti-HN1 (Rabbit mAb, Abcam, ab126705), anti-HNRNPA1 (Rabbit mAb, Abcam, ab177152), and anti-GAPDH (Mouse mAb, Abcam, ab9484) separately at room temperature for 2 hours (h), then washed with TBST buffer, and incubated with corresponding secondary antibodies conjugated with horseradish peroxidase (HRP) (Anti-rabbit IgG, Cell Signaling Technology, #7074; Anti-mouse IgG, Cell Signaling Technology, #7076) for 1 h. Then protein levels were detected by ECL (Tanon) and images were captured using an imaging system (SageCreation).

Techniques: Quantitative RT-PCR, Western Blot, Cell Counting, CCK-8 Assay, Staining, Over Expression, Activity Assay

Journal: Neurobiology of aging

Article Title: Insulin and adipokine signaling and their cross-regulation in postmortem human brain

doi: 10.1016/j.neurobiolaging.2019.08.012

Figure Lengend Snippet: Antibodies used for western blotting

Article Snippet: Ratios of phosphorylated to total levels of each antigen were calculated. table ft1 table-wrap mode="anchored" t5 caption a7 Antigen Antibody (Ab) Ab Type Ab dilution AdipoR1 Santa Cruz 518,030 Mouse mAb 1:1000 AdipoR2 Santa Cruz 514045 Mouse mAb 1:1000 AKT1–3 Santa Cruz 8312 Rabbit pAb 1:500 pS 473 AKT1–3 Santa Cruz 514032 Mouse mAb 1:750 pT 308 AKT1–3 Santa Cruz 271964 Mouse mAb 1:750 AMPKα1/2 Santa Cruz 74461 Mouse mAb 1:750 pT 183/172 AMPKα1/2 Abcam 23875 Rabbit pAb 1:1000 APPL1 Santa Cruz 271,909 Mouse mAb 1:750 IRβ Santa Cruz 81465 Mouse mAb 1:500 pY 960 IRβ Invitrogen 44–800G Rabbit pAb 1:1000 pY 1150/1151 IRβ Santa Cruz 81500 Mouse mAb 1:1000 IRS-1 Santa Cruz 8038 Mouse mAb 1:750 JAK2 Santa Cruz 390,539 Mouse mAb 1:750 pY 1007/1008 JAK2 Cell Signaling #3776 Rabbit mAb 1:1000 OB-R Santa Cruz 8391 Mouse mAb 1:750 STAT3 Santa Cruz 8019 Mouse mAb 1:750 pY 705 STAT3 Santa Cruz 8059 Mouse mAb 1:750 T-Cadherin Santa Cruz 166875 Mouse mAb 1:750 Open in a separate window Key: AdipoR1, adiponectin receptor 1; AdipoR2, adiponectin receptor 2; AMPK, adenosine monophosphate-dependent protein kinase; APPL1, adaptor protein containing pleckstrin homology domain phosphotyrosine-binding domain and leucine zipper motif; OB-R, leptin receptor; JAK2, Janus kinase 2; IR, insulin receptor; STAT3, signal transducer and activator of transcription 3; pY, phosphorylated; IRS-1, insulin receptor substrate-1.

Techniques: Western Blot