Sox10

HCT116, SW480, and HT29 cells were exposed to Ptero and/or MLT for 24 h and then were lysed in Radio-Immunoprecipitation Assay (RIPA) buffer (2 mM EDTA, 150 mM NaCl, 50 mM Tris-HCl, and 1% Triton X-100) containing protease inhibitors and phosphatase inhibitors. The protein samples were separated on 8% to 15% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and were transferred to nitrocellulose membranes. The membranes were incubated with primary antibodies of PARP (Cell signaling, #9542), cleaved-caspase-3 (Cell signaling, #9661S), NEDD9 (Cell signaling, #4044S), SOX9 (Cell signaling, #82630S), SOX10 (Cell signaling, #69661S), Bcl-xL (Cell signaling, #2764), and β-actin (Sigma, A5316). These antibodies were diluted in 3% Bovine serum albumin (BSA) and in PBS-Tween20 (1:500–1:2000) at 4 °C overnight, washed with PBS-Tween20, and then incubated with HRP-conjugated secondary antibody (Santacruz, sc-516102, sc-2357) (1:2000). The protein expression was visualized by using Enhanced chemiluminescence (ECL) Western blotting detection reagent (GE Healthcare, Amersham, UK).

The following antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA); MITF (1:1000; #12590), Her3/ERBB3 XP (1:1000; #12708), Phospho-Her3/ERBB3-Tyrosine-1289 (1:1000; #4791), FOXD3 (1:1000; #2019), SOX10 (1:1000; #14374), Akt (1:2000; #9272), Phospho-Akt-Serine-473 XP (1:2000; #4060), and Histone H3 (1:3000; #4499) was used as loading control. Secondary antibody against rabbit (1:5000; P0448) was purchased from Dako (Agilent Technologies, Glostrup, Denmark). The small molecular inhibitor vemurafenib (Plexxikon 4032) was obtained from Selleck Chemicals (Houston, TX, USA). The NRG1-beta ligand was purchased from ImmunoTools GmbH (Friesoythe, Germany). The NRG1-beta ligand was used to enhance the pERBB3 (T1289) signal, as our Phospho-Her3/ERBB3-Tyrosine-1289 antibody did not detect pERBB3 (T1289) levels without NRG1-beta addition (see also Supplementary Figure S3A-S3B). The reason for choosing 15min of incubation time and a NRG1-beta ligand concentration of 10ng/ml was due to a previous study where they showed efficient activation of the ERBB3 receptor in the WM115 cell line under these conditions [18 (link)].

Antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA); MITF (1:1000; #12590), Her3/ERBB3 XP (1:1000; #12708), AXL (1:1000 #8661), SOX10 (1:1000; #14374), Phospho‐Akt‐Serine‐473 XP® (1:2000; #4060), Phospho‐p44/42 (ERK1/2) XP® (1:2000; #4370S) and Histone H3 (1:3000; #4499) was used as a loading control. Secondary antibody against rabbit (1:5000; P0448) was purchased from Dako (Agilent Technologies, Glostrup, Denmark). The small molecular inhibitors vemurafenib (Plexxikon 4032), AZD8931 (Sapatinib) and R428 (BGB324/bemcentinib) were obtained from Selleck Chemicals (Houston, TX, USA). Recombinant human NRG1‐beta protein‐ligand was purchased from ImmunoTools GmbH (Friesoythe, Germany). Recombinant human GAS6 protein‐ligand was purchased from R&D Systems (Minneapolis, USA).

For histology and IHC, mouse brains were processed, immunostained, and quantitatively analyzed as previously described^{10 (link),20 (link),53} . For western blot, samples were snap-frozen and lysed, blotted, and stained as previously described⁵³ . Primary antibodies used were: cC3 1:100 (Biocare, #229), pRB diluted x (x), OLIG2 diluted 1:100 (Cell Marque, # 387R-14), pOLIG2 diluted 1:3000 (x), SOX10 diluted 1:200 (Cell Signaling Technology, #7833S), MHC Class II glycoprotein H2-Ea diluted 1:200 (Novus, # NBP1-43312), IBA1 1:2000 (Wako Chemicals, #019-19741) and HLA-DR diluted 1:50 (Wako NBP2-47670), and p4EBP1 diluted 1:500. HLA-DR studies were performed as previously described^{51 (link)}. All other stained slides were counterstained with DAPI, digitally imaged using an Aperio Scan Scope XT (Aperio), and subjected to automated cell counting using Tissue Studio (Definiens).

Transverse cryosections of 10 μm were made using a cryostat and every fifteenth or twentieth embryonic section were deposited on the same glass slide (~10 sections/slide), separating each section by 150 or 200 μm, respectively.
For immunofluorescence (IF) analyses embryos sections were fixed in 4% paraformaldehyde for 5 min, then washed in PBS (3 × 5 min, same for all following washes) and treated for permeabilization with 0.5% Triton X-100 in PBS for 5 min. After washes, slides were incubated with a blocking solution (5% Goat serum, 0.1%Triton X-100 in PBS) for 45 min at RT, and then overnight at 4°C with primary antibodies diluted in PBS. The next day, after washes, corresponding secondary antibodies (diluted at 1/500 in PBS) were added for 1 h at RT, and then incubated with fluorescent mounting medium for 30 min (Dako, 1/5000). The following primary antibodies were used to detect Phox2b (Santa Cruz Biotechnology, #sc-376997, 1/100), Sox10 (Cell Signaling Technology, #89356S, 1/200); Ki67 (Abcam, #ab 15580, 1/200), TH (Abcam, #ab 76442, 1/500), βIII-tubulin (Cell Signaling Technology, #5568, 1/200), ALK (Cell Signaling, D5F3® #3633, 1/100); and revealed using the secondary Ab: Goat-a-Mouse-Alexa Fluor® 488 (ThermoFisher scientific, #A-11001), Goat-a-Rabbit- Alexa Fluor ® 647 (ThermoFisher scientific, #A-21246), Goat-a-Chicken-Alexa Fluor® 647 (Abcam, #ab 150175).

Neural crest protocol was adapted from Hackland et al.^{24 (link)}. Cells were harvested and plated at 40,000 cells/cm² on wells coated with Geltrex (Thermofisher) in neural crest medium (DMEM/F12 basal, Sigma, 1× N2 supplement, Thermofisher, 1× Non-essential Amino Acids, Thermofisher, 1× GlutaMax, Thermofisher, 2 µM SB431542, Tocris, 1 µM CHIR99021, Tocris, 1 µM DMH1, Tocris and BMP4 15 ng/ml, Gibco). Cells were plated either with 10 µM of Rock Inhibitor (Y-27632, Generon) or without Rock Inhibitor and 1 µM of IWP-2 (Tocris) for the first 48 h. Plates were placed in an incubator at 37 °C and 5%CO₂. Medium was replaced after 48 h with fresh neural crest medium. Medium was replaced every other day. Cells were fixed on the 5th day of neural crest differentiation and stained for SOX10 (Cell Signalling Technology, 89356S, 1:500).