rat recombinant dkk1 rdkk1  (R&D Systems)

Journal: Nature Communications

Article Title: CCN1 interacts with integrins to regulate intestinal stem cell proliferation and differentiation

doi: 10.1038/s41467-022-30851-1

Figure Lengend Snippet: Ccn1 Δ Lgr5 mice were analyzed at day 5-post corn oil or TM. a Immunofluorescence staining of Notch1 intracellular domain (N1-ICD, red) in the crypt of the jejunum. DAPI is counterstaining. Representative images are shown from three independent replicates. Bars: 20 µm. b Immunoblot analysis of indicated protein expression (N1-ICD, N2-ICD, Jag1, DLL1, and DLL2) in the jejunum. β-actin as a loading control. Representative images are shown from at least three replicates. c qPCR analysis of Notch target genes ( Hes1, Hey1 , and HeyL ) and Atoh1 . Data represent the mean ± SEM of four biological replicates. One-sided t -test. *** p < 0.001. d Immunofluorescence staining of active β-catenin in the crypt of Ccn1 Δ Lgr5 mice. Images are representatives of three independent replicates. Bars: 20 µm. e Immunoblot analysis of active and total β-catenin. Images are representative of three replicates. f , g qPCR analysis of Wnt target genes ( Axin2, Cd44, Ephb2 , and Myc ; f ), Wnt ligands ( Wnt3a, Wnt5a , and Wnt3b ; g ), and a secreted Wnt antagonist Dkk1 ( g ). Data represent the mean ± SEM of four biological replicates. One-sided t -test. * p < 0.05; ** p < 0.01; *** p < 0.001. Source data and the exact p -values are provided in a Source Data file.

Article Snippet: Recombinant Jag1 protein (599-JG) and Dkk1 protein (5897-DK) were from R&D systems.

Techniques: Immunofluorescence, Staining, Western Blot, Expressing, Control

Journal: Nature Communications

Article Title: CCN1 interacts with integrins to regulate intestinal stem cell proliferation and differentiation

doi: 10.1038/s41467-022-30851-1

Figure Lengend Snippet: a AP staining and immunofluorescence staining of markers (Lyz1, Muc2, Lgr5 + GFP, and Ki67) for the analysis of differentiation and proliferation of the Ccn1 Δ Lgr5 organoids. Ccn1 Δ Lgr5 organoids were treated with vehicle or 4-OHT (1 µM) for 5 days. CCN1 (4 µg per ml) was added to organoids 1 day after 4-OHT. Inhibitors (Dasatinib, 10 nM; Super-TDU, 50 nM) were pretreated 30 min before CCN1. Representative images are shown from three independent replicates. Bars: 100 µm. b qPCR analysis of lineage markers in organoids treated as in a . Data represent the mean ± SEM of four biological replicates. One-sided t -test. * p < 0.05; ** p < 0.01; *** p < 0.001; ns not significant. c Immunoblot analysis of indicated proteins from Ccn1 Δ Lgr5 organoids treated as described in a . Representative images are shown from three replicates. d qPCR analysis of Wnt ligands ( Wnt3a, Wnt5a, Wnt5b , and Wnt9b ) and Wnt antagonists ( Dkk1, Dkk2 , and sFRP5 ) in Ccn1 Δ Lgr5 organoids treated with vehicle or 4-OHT (1 µM, d5). Data represent the mean ± SEM of four biological replicates. One-sided t -test. *** p < 0.001; ns not significant. e qPCR analysis of Dkk1 expression in Ccn1 Δ Lgr5 organoids treated as indicated (CCN1, 4 µg per ml; Super-TDU, 50 nM). Data represent the mean ± SEM of four biological replicates. One-sided t -test. ** p < 0.01; ns not significant. f Immunoblot analysis of active and total β-catenin in Ccn1 Δ Lgr5 organoids treated as indicated (CCN1, 4 µg per ml ; Super-TDU, 10 and 50 nM). β-actin as a loading control. Representative images are shown from three replicates. g qPCR analysis of Wnt target genes ( Axin2, Cd44, and Ephb2 ) in Ccn1 Δ Lgr5 organoids treated as indicated. Data represent the mean ± SEM of four biological replicates. One-sided t -test. * p < 0.05; ** p < 0.01; *** p < 0.001. h Immunofluorescence staining of Lgr5 + GFP and Ki67 in Ccn1 Δ Lgr5 organoids treated as indicated. A neutralizing anti-Dkk1 antibody (5 µg per ml) or isotype control IgG was treated in place of inhibitors as in a . Representative images are shown from three replicates. Bars: 100 µm. Source data and the exact p -values are provided in a Source Data file.

Article Snippet: Recombinant Jag1 protein (599-JG) and Dkk1 protein (5897-DK) were from R&D systems.

Techniques: Staining, Immunofluorescence, Western Blot, Expressing, Control

Journal: Nature Communications

Article Title: CCN1 interacts with integrins to regulate intestinal stem cell proliferation and differentiation

doi: 10.1038/s41467-022-30851-1

Figure Lengend Snippet: a , b Yap flox/+ ; Lgr5 CreERT or Yap flox/flox ; Lgr5 CreERT ( Yap Δ Lgr5 ) mice treated with corn oil or TM (day 5, n = 4 each). a Immunofluorescence staining of YAP, CCN1, and lineage markers ( Lgr5 + GFP, Lyz1, and Muc2), and AP staining on the jejunum sections. Images are representative of three independent replicates. Bars: 20 µm. b qPCR analysis of indicated genes. Data represent the mean ± SEM of four biological replicates. One-sided t -test. * p < 0.05; ** p < 0.01; *** p < 0.001. c – e Yap Δ Lgr5 organoids were treated with 4-OHT (1 µM), followed by CCN1 protein (4 µg per ml). c AP staining and immunofluorescence staining of Lyz1, Muc2, Lgr5 + GFP, and Ki67. DAPI is counterstaining. Images are representative of three replicates. Bars: 100 µm. d qPCR analysis of indicated genes. Data represent the mean ± SEM of four biological replicates. One-sided t -test. ** p < 0.01; *** p < 0.001; ns not significant. e Immunoblot analysis of indicated proteins. Representative images are shown from three replicates. f , g Yap Δ Lgr5 organoids were treated with 4-OHT (1 µM), followed by Dkk1 proteins (50 ng per ml). f Immunofluorescence staining of Lgr5 + GFP and Ki67. DAPI is counterstaining. Representative images are shown from three independent replicates. Bar: 100 µm. g qPCR analysis on Axin2, Lgr5, Lyz1 , and Muc2 . Data represent the mean ± SEM of four biological replicates. One-sided t -test. ** p < 0.01; *** p < 0.001. Source data and the exac t p -values are provided in a Source Data file.

Article Snippet: Recombinant Jag1 protein (599-JG) and Dkk1 protein (5897-DK) were from R&D systems.

Techniques: Immunofluorescence, Staining, Western Blot

Journal: Nature Communications

Article Title: CCN1 interacts with integrins to regulate intestinal stem cell proliferation and differentiation

doi: 10.1038/s41467-022-30851-1

Figure Lengend Snippet: CCN1 expressed at the luminal side of the crypt base binds to integrins α v β 3 /α v β 5 on, presumably, Paneth cells and Lgr5 + ISCs, which elicits two distinct signaling pathways bifurcating downstream of FAK. For the regulation of Wnt signaling, FAK recruits and activates Src, leading to YAP stabilization and activation through phosphorylation at tyrosine 357. Active p-YAP (Y357) can enter the nucleus, where it promotes TEADs-dependent transcription of target genes, such as a secreted Wnt antagonist Dkk1 , which interferes with Wnt binding to its cognate receptor Frizzled by sequestering a Wnt co-receptor LRP5/6. Concurrently, YAP-TEADs can further increase the expression of another target gene Ccn1 in the crypt, suggesting a regulatory loop between CCN1 and YAP. On the other hand, integrin-FAK signaling results in NF-κB activation and subsequent induction of Jag1, a Notch ligand, which promotes Notch signaling important for ISC fate decision in the crypt base.

Article Snippet: Recombinant Jag1 protein (599-JG) and Dkk1 protein (5897-DK) were from R&D systems.

Techniques: Protein-Protein interactions, Activation Assay, Phospho-proteomics, Binding Assay, Expressing

Journal: Nature medicine

Article Title: DKK2 imparts tumor immunity evasion through β-catenin-independent suppression of cytotoxic immune cell activation

doi: 10.1038/nm.4496

Figure Lengend Snippet: (a-c) . Littermates of Apc Min/+ and Apc Min/+ Dkk2 −/− mice were housed for 20 weeks (female) or 22 weeks (male), and their polyps numbers in intestines were counted and measured under a stereomicroscope after staining with methylene-blue (two-tailed Student t-Test for polyp number and two-way Anova for size; n=7). Representative H&E staining images of intestinal sections from an Apc Min/+ mouse and its Apc Min/+ Dkk2 −/− littermates are shown. (d ) Binding of the anti-DKK2 antibody 5F8 to mouse DKK2 and DKK1 proteins in an ELISA assay(n=3). (e) HEK293 cells were transfected with the Wnt reporter gene TOPFlash and treated with WNT3A conditioned medium (CM), DKK2 CM or DKK1 CM, and 5F8 (18 µg/ml) as indicated (one-way Anova; n=3). (f) HEK293 cells were transfected with LacZ (a control) or LRP5 expression plasmid. The binding of DKK2-AP fusion protein to the cells in the presence or absence of 5F8 (18 µg/ml) were determined (Two-way Anova; n=3). (g) Mice (16 weeks old male) were treated with 5F8 and an isotype antibody (IgG3) (8 mg/kg, once a week, i.p.) and their survival was recorded (P=0.004; Two-sided Mantel-Cox Log-Rank test, n=9). Data are presented as means±SEM (a-b) and ±sd (d-f) with P values shown, and experiments were repeated twice (d-f).

Article Snippet: Recombinant mouse DKK2 or DKK1 protein (20 ng/ml, R&D) in a blocking buffer (1% BSA in PBS) was incubated in a 384-well microtiter plate for overnight at 4°C.

Techniques: Staining, Two Tailed Test, Binding Assay, Enzyme-linked Immunosorbent Assay, Transfection, Expressing, Plasmid Preparation