Figure 4. cNICD levels in the chick PSM are elevated after exposure to XAV939, Roscovitine, DRB or PHA767491.
(A): A polyclonal cNICD antibody was raised against the N-term sequence of the cleaved chicken Notch1 intracellular domain. The epitope is only exposed after gamma secretase cleavage and is not accessible in the uncleaved form. By Western Blot analysis the antibody detects a band of protein at around 90KDa (see arrow) in an in vitro-generated recombinant cNICD sample, which is not detected in the control IVT sample; this band disappears in half chick PSM pools (see ‘Materials and methods’) after 3 hr treatment with 150 nM LY411575, a concentration shown previously to have no toxic effects to PSM tissue (Bone et al., 2014). (B): Immunohistochemistry for cNICD protein on 16 μm sagittal sections of sucrose agar embedded HH10 chicken embryo tails showing dynamic phases of localisation in the PSM and schematic representations to the right of each panel. S1 - S3 = somite; S0 - S-1 = prospective somite region of anterior PSM. (C): A schematic illustrating the cNICD degradation assay: corresponding pools of 9 PSM explants are incubated in the presence or absence of an inhibitor for 3 (chick) or 4 hr (mouse), before treating all pools with the protein synthesis inhibitor cycloheximide (CHX) for a further hour. Lysates are then collected for Western Blot analysis. (D): Representative Western Blot showing levels of cNICD protein in lysates of chick PSM pools are increased after treatment with XAV939, Roscovitine, DRB and PHA767491, but not with cyclopamine. Lanes 1 and 2 show exposure to 150 nM LY411575 alone for 3 hr removes all NICD and serves as a control for the western. Lanes 3 and 4 show exposure to CHX alone in the last hour of culture severely depletes NICD levels as compared to NICD levels in the pool of contralateral PSM explants cultured in DMSO. (E): Levels of mNICD in the mouse PSM pools are increased following treatment with XAV939. Lanes 1 and 2 show that NICD protein levels drop drastically after exposure for 1h to CHX as compared to strong NICD signal in lysate from the contralateral half PSMs treated with EtOH. (F): Representative Western Blot showing levels of cNICD protein in lysates of chick PSM pools are increased after treatment with XAV939, Roscovitine and DRB when LY411575 is added to the culture for the last hour (in place of CHX). Lanes 1 and 2 show exposure to LY411575 alone only in the last hour severely depletes NICD levels as compared to the NICD levels in the pool of contralateral PSM explants cultured in DMSO.
Figure 4—figure supplement 1. XAV939, Roscovitine, and DRB treatment increases the level of cNICD in the chick PSM.
