Figure 4.

Casitas B-lineage lymphoma (c-Cbl)–Y371H is a Wnt inducer that up-regulates β-catenin. A: Cell lysates of HT-29 cells co-expressing hemagglutinin (HA)–tagged c-Cbl wild-type (WT) or c-Cbl–Y371H and the TCF-responsive promoter reporter pBARLS and nonresponsive control reporter pfuBARLS tethered to luciferase reporter were analyzed for luciferase assay.²⁶ Activity of the Wnt signaling pathway was quantified by measuring relative firefly luciferase units normalized to protein concentration. Luciferase activity was noted in the presence of c-Cbl-Y371H, whereas c-Cbl WT suppresses Wnt activity. B: Hs:Wnt-8–enhanced green fluorescent protein (eGFP) transgenic zebrafish embryos were heat shocked at 38°C for 20 minutes at 10 hours post fertilization (hpf) and imaged for 24 hpf. C: Ten deyolked control and heat shocked embryos were lyzed at 36 hpf and investigated for β-catenin. Tubulin served as a loading control. D: Hs:Wnt-8–eGFP transgenic zebrafish embryos were injected with capped mRNA of Myc-tagged c-Cbl and Myc-tagged c-Cbl–Y371H at one cell stage and then heat shocked. Ten deyolked eGFP-positive embryos harvested at 36 hpf were investigated for β-catenin and tubulin. Equal amounts of lysates were investigated separately with anti-Myc–tagged antibody. E: Primary human umbilical venin endothelial cells (HUVECs) co-transfected with c-Cbl–Y371H construct and control and β-catenin siRNA (si) oligos were serum starved overnight followed by stimulation with 50 ng/mL of Wnt-3a ligand. The media of endothelial cells collected after 24 hours of Wnt treatment were analyzed for vascular endothelial growth factor (VEGF) levels. F: HUVECs co-expressing control (CTL) or HA-tagged c-Cbl– Y371H and control and β-catenin si oligos were serum starved and treated with 50 ng/mL of Wnt-3a. Cells were then seeded in a 96-well plate coated with Matrigel and analyzed for tube formation within 24 hours. G: The tube lengths were measured in the Matrigel with ImageJ version 1.43t. H: Fli-eGFP transgenic zebrafish two-cell-stage embryos injected with different mRNA. LacZ-injected embryos served as controls. Representative images from randomly selected fish from total injected at 24 hpf are shown (Table 3). The dashed white line represents the junction of main body and tail, after which the tail vessels are measured (white dotted line). The yellow brackets indicate the caudal vessel plexus. I: Mean length of tail vessels of 10 randomly selected fish from total injected. The images under same light exposure settings were obtained from 10 randomly selected fish per group from total live fish and analyzed for the length of the tail vessels. The tail vessels were marked from the junction of the body and the tail (white dotted line in H) going caudally using Image-Pro and averaged per group.9, 18J: The images of fish under same light exposure settings were obtained for 10 randomly selected fish per group from total live fish. The region of interest was marked and analyzed for the intensity of caudal vessel plexus using ImageJ. Mean intensity is shown. Data are expressed as means ± SEM (A, E, G, I, and J). n = 3 (A, C, D, F, H–J); n = 62 heat shocked and 80 control embryos (B); n = 2 (G, six images from two separate experiments performed in triplicate). ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 versus control (t-test with Bonferroni correction). Scale bars = 100 μm. Original magnification, ×5 (B and H).