Journal: Oncotarget

Article Title: Positive feedback loop of hepatoma-derived growth factor and β-catenin promotes carcinogenesis of colorectal cancer

doi:

Figure Lengend Snippet: A. - B. β-catenin knockdown significantly suppressed HDGF mRNA expression in HCT116 A. and HT29 B. cells by real-time PCR analysis, respectively; C. - E. β-catenin knockdown inhibited HDGF protein expression in HCT116 cells C. and mainly inhibited nuclear HDGF, β-catenin, c-Myc, cyclin D1, MMP9 and phos-GSK-3β (Ser9) protein expression in HCT116 D. and HT29 E. cells; F. Recombinant Wnt3a and DKK1 increased and decreased HDGF and β-catenin expression in HCT116 cells, respectively; G. - I. Recombinant Wnt3a and DKK1 increased and decreased nuclear and cytoplasmic HDGF, β-catenin, c-Myc, cyclin D1, MMP9 and phos-GSK-3β (Ser9) protein expression in HCT116 G. , I. and LOVO H. cells by Western blot analysis, respectively.

Article Snippet: To further verify the effect of β-catenin on HDGF expression in CRC cells, HDGF and β-catenin protein expressions in HCT116 were induced by 100ng/ml human recombinant Wnt3a (R&D SYSTEMS) and inhibited by 200ng/ml human recombinant DKK1 (R&D SYSTEMS) for 48 hours by Western blot analysis, respectively (Figure ).

Techniques: Knockdown, Expressing, Real-time Polymerase Chain Reaction, Recombinant, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Calreticulin regulates a switch between osteoblast and chondrocyte lineages derived from murine embryonic stem cells

doi: 10.1074/jbc.RA119.011029

Figure Lengend Snippet: Recombinant DKK1 increases chondrocyte specification while blocking osteoblast specification. Differentiated WT ESCs were stained with either Alcian blue or von Kossa as indicated ( A ), treated without or with rbDKK1 for days 6–17 of osteoblast differentiation. B , RT-qPCR analysis of chondrocyte or osteoblast markers in WT ESCs differentiated in the absence (■) or presence (▿) of rbDKK1 for days 6–17. C , Western blot analysis of whole-cell lysates harvested at the indicated times from ESCs during osteoblast differentiation in the absence (■, WT ESCs; ♦, crt −/− ESCs) or presence of mouse rbDKK1 for days 6–17 (▿, WT ESCs; ♢, crt −/− ESCs). Utilized antibodies were specific to SOX9 and GAPDH as an internal control. The graph is a quantitative representation of the density of the Western blotting bands of SOX9 normalized to internal control GAPDH. Data are expressed as means ± S.D. ( error bars ), n ≥ 3; two-way ANOVA of transcript levels ( B ) and band densities ( C ). B , Sox9 , p < 0.0001, F = 31.25; aggrecan, p < 0.0001, F = 94.4; osterix, p = 0.0073, F = 7.874; osteocalcin, p = 0.0007, F = 13.24. C , p < 0.0001 and F = 24.55 for WT control and crt −/− ESCs data set; p < 0.0001 and F = 48.83 for WT control ESCs treated or not with rbDKK1; p = 0.3412 and F = 0.9386 for crt −/− ESCs treated or not with rbDKK1. Bonferroni post hoc test was as indicated: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ψ, p < 0.001 for WT control ESCs treated or not with rbDKK1; ***, p < 0.001 for WT control and crt −/− ESCs ( C ). D , confocal images of immunolocalization using SOX9, type II collagen, or osterix antibodies on WT ESCs at day 19 of the osteogenic differentiation protocol in the presence of rbDKK1 on days 6–17. Dual-channel grayscale images of a single field are displayed with the labeled protein of interest in the left panel and DAPI-stained nuclei in the right panel , and the RGB panel is a merged image of green (protein of interest) and blue (DAPI). See E for the WT ESC nontreated control. Scale bar , 50 μm.

Article Snippet: Differentiating cells were treated with the inhibitor of intranuclear transport of NFAT A-285222 ( M r 416.28) ( ) (a generous gift from AbbVie) at 5 μg/ml, a 3 μ m concentration of the GSK inhibitor CHIR99021 (Tocris), or 100 ng/ml mouse recombinant DKK1 (R&D Systems), each continuously for days 6–17.

Techniques: Recombinant, Blocking Assay, Staining, Quantitative RT-PCR, Western Blot, Labeling

Journal: Cell reports

Article Title: Complementary Wnt Sources Regulate Lymphatic Vascular Development via PROX1-Dependent Wnt/β-Catenin Signaling

doi: 10.1016/j.celrep.2018.09.049

Figure Lengend Snippet: (A) HLECs were exposed to oscillatory shear stress (OSS) for the indicated number of hours. Subsequently, the cells were harvested and analyzed by western blotting using the indicated antibodies. Phosphorylation of LRP6 is increased by OSS. (B and C) HLECs were cultured under OSS with recombinant DKK1, which inhibits the interaction between Wnt ligands and LRP co-receptors (B), or LGK-974, which inhibits the secretion of Wnt ligands from cells (C). Subsequently, RNA was extracted, and the expression of AXIN2 , FOXC2 , and GATA2 was evaluated by real-time qPCR analysis. The data were normalized to GAPDH . Both DKK1 and LGK-974 inhibit OSS-induced expression of AXIN2, FOXC2 , and GATA2 . (D and E) E15.5 LVVs are observed in both Lyve1Cre ; Wls f/f (E) and control (D) littermate embryos (arrows). (F–I) Whole-mount immunohistochemistry on the dorsal skin of E16.5 wild-type (F) and Lyve1Cre;Wls f/f (G) embryos reveals normal lymphatic vessels. The diameters of lymphatic vessels (H) and their migration toward the dorsal midline (I) are not different between E17.5 control and mutant embryos. (J–L)Normal-looking LVs are observed inmesenteric lymphatic vessels of P2 control (J, arrow) and Lyve1Cre;Wls f/f mice (K,arrow). The number of E18.5 LVs is not changed in Lyve1-Cre;Wls f/f mice (L). LVV, lymphovenous valve; LV, lymphatic valve;IJV, internal jugular vein; SCV, subclavian vein. Scale bars, 100 mm (D–G) and 200 mm (J and K). Statistics: (A)–(C), n = 3; (D)–(L), n = 4 per genotype per stage; **p < 0.01, ***p < 0.001, ****p < 0.0001. Error bars in graphs represent ± SEM.

Article Snippet: Recombinant Wnt3a and recombinant DKK1 were purchased from R&D systems.

Techniques: Shear, Western Blot, Cell Culture, Recombinant, Expressing, Control, Immunohistochemistry, Migration, Mutagenesis