Sc 166962

Injected embryos were sorted by fluorescence dissecting scope-detectable notochord fluorescence (indicating the presence of the UAS-transgene) and imaged at 5 dpf. Animals with strong fluorescence were fixed in 4% paraformaldehyde at 4°C overnight. Subsequently, embryos were embedded in paraffin, sectioned at 5 µm, deparaffinated and stained with Hematoxylin and Eosin (H&E) according to standard protocols. Immunohistochemical studies were performed according to the manufacturer's protocol using anti-pERK (4376, Cell Signaling Technology, 1:500), anti-Cytokeratin (961, Abcam, 1:100), anti-TP53 (GTX128135, GeneTex, 1:200), anti-Tbxta (a gift from Andy Oates, École polytechnique fédérale de Lausanne; Webb et al., 2016 (link)) and anti-Brachyury (sc-20109 or sc-166962; Santa Cruz, 1:200). Work on chordoma sections was performed under approval by BASEC-no. 2017-00017 by the Cantonal Ethics Commission Zürich. Live animals were imaged using a Leica M205FA stereo microscope. Histological sections were imaged using a Zeiss Axioscan Z1 Slidescanner using a Plan Apochromat 20× objective.

Antibodies against octamer-binding transcription factor 4 (1:1000, Oct-4; ab200834, abcam), Brachyury (1:1000, sc-166962, Santa Cruz Biotechnology), GATA-binding protein 4 (1:1000, GATA4; sc-25310, Santa Cruz Biotechnology), SRY box transcription factor 17 (1:1000, Sox17; AF1924, R&D system), neuronal differentiation 1 (1:1000, NeuroD1; ab205300, abcam), XRCC5 (1:2000, AF5819, R&D system), IGF2BP1 (1:1000, ab184305, abcam), RBBP4 (1:1000, MAB7416, R&D system), NPM1 (1:1000, sc-271737, Santa Cruz Biotechnology), PCNA (1:1000, sc-56, Santa Cruz Biotechnology), GNAI2 (1:1000, ab137050, abcam), PHB2 (1:1000, sc-133084, Santa Cruz Biotechnology), LAMB1 (1:1000, sc-17810, Santa Cruz Biotechnology), LAMC1 (1:1000, sc-17751, Santa Cruz Biotechnology), β-catenin (1:1000, sc-7963, Santa Cruz Biotechnology), AKT (1:2000, #4691, Cell Signaling Technology), Jun amino-terminal kinases (JNK) (1:1000, #9252, Cell Signaling Technology), phosphorylated (p)-JNK (1:1000, #9255, Cell Signaling Technology), p38 (1:1000, sc-81621, Santa Cruz Biotechnology), p-p38 (1:1000, #9216, Cell Signaling Technology), TGF-β (1:1000, #3711, Cell Signaling Technology), CTGF (1:1000, sc-385970, Santa Cruz Biotechnology), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; (1:3000, sc-47724, Santa Cruz Biotechnology) were employed for the western blot analysis.

The antibodies and reagents used in this experiment are listed in Table 1.Table 1

List of the antibodies and reagents

Product	Catalog number	Manufacturer
Antibody
Aggrecan (rabbit, polyclonal)	ab36861	Abcam (Cambridge, UK)
Tie 2 (goat, polyclonal)	AF762	R&D systems, USA
Brachyury (mouse, monoclonal)	sc-166962	Santa Cruz, USA
Collagen type II (rabbit, polyclonal)	ab34712	Abcam (Cambridge, UK)
Collagen type I (rabbit, monoclonal)	ab270993	Abcam (Cambridge, UK)
CGRP (mouse, monoclonal)	ab81887	Abcam (Cambridge, UK)
MMP-13 (rabbit, polyclonal)	ab39012	Abcam (Cambridge, UK)
IL-1β (goat, polyclonal)	AF-501-NA	Novus Biologicals, USA
TNF-α (rabbit, polyclonal)	ab6671	Abcam (Cambridge, UK)
Staining
4% paraformaldehyde phosphate buffer solution	PC2031-100–00	Biosesang, South Korea
Alexa Fluor® 488 secondary antibody	A11034	Invitrogen, USA
Alexa Fluor® 488 secondary antibody	A11029	Invitrogen, USA
Alexa Fluor® 568 secondary antibody	A10042	Invitrogen, USA
Alexa Fluor® 647 secondary antibody	A21469	Invitrogen, USA
DAPI	D1306	Invitrogen, USA

CGRP Calcitonin-gene related peptide, MMP-13 Matrix metalloproteinase-13, IL-1β Interleukin-1β, TNF- α Tumor necrosis factor alpha, DAPI Diamidino-2-phenylinodole