Functional validation analyses

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), phosphate-buffered saline, glutamine and antibiotics were obtained from Euroclone (Wetherby, UK). Fugene6 transfection reagent and complete protease inhibitor cocktail tablets were purchased from Roche Diagnostics (Mannheim, Germany). CHX and MG132 were from Sigma-Aldridge (St. Louis, MO, USA). Trans-Blot Turbo Transfer Packs were from Bio-Rad Laboratories (Hercules, CA, USA). ECL Western Blotting Detection reagents were obtained from Pierce Biotechnology (Rockford, IL, USA). QuiKChange II Site-Directed Mutagenesis kit was obtained from Stratagene (La Jolla, CA, USA). pcDNA6.2/V5-HisA eukaryotic expression vector and EGF were from Invitrogen (Carlsbad, CA, USA). Protein G Sepharose was obtained from GE Healthcare (Freiburg, Germany). The following antibodies were used: mouse monoclonal anti-V5 (Invitrogen); mouse monoclonal anti-Myc, rabbit polyclonal anti-Na/K-ATPase, rabbit polyclonal anti-p44/42 MAPK1/3, mouse monoclonal anti-phospho-p44/42 MAPK1/3 (Thr202/Tyr204 of Erk1 and Thr185/Tyr187 of Erk2) (Cell Signaling Technology, Danvers, MA, USA); mouse monoclonal anti-β-actin, mouse monoclonal anti-HSP90, horseradish peroxidase-conjugated anti-rabbit or anti-mouse (Sigma-Aldrich); mouse monoclonal anti-GAPDH, rabbit polyclonal anti-YY1 (Santa Cruz, Dallas, CA, USA); goat anti-mouse conjugated to Alexa Fluor 594 and Alexa Fluor 488 phalloidin dye were from Molecular Probes (Eugene, OR, USA). Vectashield antifade medium containing DAPI was purchased from Vector Laboratories (Burlingame, CA, USA). Phenylthiourea (PTU), Alcian blue and MS-222 were obtained from Sigma-Aldrich. mMESSAGE SP6 Transcription Kit was from TermoFisher Scientific (Waltham, MA, USA), while the Q5 Site-Directed Mutagenesis Kit was from New England Biolabs (Ipswich, MA, USA). The myl7-specific probe was previously described (22).

The entire coding sequence of WT SHOC2 was cloned into the pcDNA6.2/V5-HisA eukaryotic expression vector. Mutant SHOC2 constructs carrying the p.Gly53Arg, p.Met173Val, p.Gln269Arg, p.Thr411Ala or p.Met173_Leu174delinsIlePhe amino acid substitutions were generated by PCR-based site-directed mutagenesis using the QuiKChange II Site-Directed Mutagenesis Kit (15). The identity of each construct was verified by bidirectional sequencing (ABI BigDye terminator Sequencing Kit v3.1, SeqStudio Genetic Analyzer; Applied Biosystems, Foster City, CA, USA). pcDNA3.1-Myc-MRAS and pcDNA3.1-Myc-PPP1CB constructs were produced as previously reported (12). pCR-Blunt II-TOPO vector was from TermoFisher Scientific.

Zebrafish Shoc2 was amplified from cDNA of 5 days old zebrafish embryos by nested PCR using the primers pair 5′-CGAACAAAATACAAGAGCGAGAG-3′ and 5′-GCCAATCTAGATCAGACCATGGC-3′ in the first reaction and primer pair 5′-GCGGATCCATGAGCAGTACTCTG-3′ and 5′-GCCAATCTAGATCAAGCGTAATCTGGAACATCGTATGGGTAGACCATGGCGCGGTAGGG-3′ in the second reaction. In the second reaction, the HA tag was added to the C-terminus of the construct. PCR product was cloned into the pCR-Blunt II-TOPO vector to generate pCR-Blunt II-TOPO-SHOC2-HA. From here SHOC2-HA was subcloned into the BamHI and XbaI restriction sites of pCS2+ vector to generate pCS2+ SHOC2-HA. pCS2+ SHOC2-HA plasmid served as template to introduce the missense changes resulting in the S2G, G32R, M152I, M152V and T390A amino acid substitutions, which correspond to the patient-associated S2G, G53R, M173I, M173V and T411A substitutions, respectively. S2G mutation was introduced by PCR using the primer pair 5′-GCGGATCCATGGGCAGTACTCTG-3′ and 5′-GCCAATCTAGATCAAGCGTAATCTGGAACATCGTATGGGTAGACCATGGCGCGGTAGGG-3′. Other mutations were introduced by Q5 Site-Directed Mutagenesis Kit. All constructs were verified by sequencing (Macrogen Europe B.V., Amsterdam, the Netherlands). mRNA used for injections of zebrafish embryos was produced from the pCS2+ SHOC2 constructs using the mMESSAGE mMACHINE SP6 Transcription Kit.

COS-1 and Neuro2A cells were cultured in DMEM medium supplemented with 10% heat-inactivated FBS, 1% L-glutamine and antibiotics (37°C, humidified atmosphere containing 5% CO₂). Subconfluent cells were transfected using the Fugene6 transfection reagent, according to the manufacturer’s instructions. Cells were treated with CHX (100 μg/ml) or MG132 (50 μM) to analyze protein stability and degradation. Serum-free DMEM and EGF (30 ng/ml) were utilized to starve and stimulate cells, respectively.

Cells were lysed in radio-immune precipitation assay buffer, pH 8.0, supplemented with 20 mm NaF, 1 mm Na₃VO₄ and protease inhibitors. Lysates were centrifuged at 16 000 g for 20 min at 4°C, and supernatant protein concentration was determined by BCA assay (44), using BSA as standard. Western blotting (WB) and densitometric analyses were performed as previously described (15,30,33). Cell fractionation was performed as previously reported (15,33), using transfected COS-1 cells serum-starved overnight and then EGF-stimulated or left untreated. Each subcellular fraction was subjected to SDS-PAGE and WB to determine the SHOC2 distribution in the different cellular compartments. Membranes were probed with antibodies to HSP90 (cytoplasmic marker), YY1 (nuclear marker) and Na/K-ATPase (plasma membrane marker). SHOC2/MRAS and SHOC2/PPP1CB co-IP assays were performed on transfected Neuro2A cells serum starved for 16 h, and then stimulated with EGF, as reported (9,12,45).

COS-1 cells (15 × 10³) were seeded on glass coverslips, transfected with the various constructs (24 h), serum starved (16 h) and stimulated with EGF (15 min) or left unstimulated. Cells were fixed with 4% paraformaldehyde (30 min, 4°C) and permeabilized (0.05% Triton X-100, 10 min, room temperature). Cells were then stained with a mouse monoclonal anti-V5 followed by goat anti-mouse Alexa Fluor 594, and then with Alexa Fluor 488 phalloidin dye. Finally, glass coverslips were mounted on microscope slides using Vectashield antifade medium containing DAPI, and analyzed using an Olympus FluoView FV1000 apparatus, utilizing excitation spectral laser lines at 405, 488 and 568 nm. Signals from different fluorescent probes were taken in sequential scanning mode.

Experimental animal procedures were approved by the local animal experiments committee of the Royal Netherlands Academy of Arts and Sciences (KNAW), and performed following local guidelines and policies in agreement with national and European law. Maintenance of zebrafish was performed as described previously (46,47). PTU (0.003%, v/v) was added to the E3 medium at 24 hpf to block pigmentation.

Zebrafish embryos were injected at the one-cell stage in the yolk with a mixture of 40 ng/μl mRNA encoding SHOC2 (WT or variant), and 50 ng/μl mRNA encoding GFP. GFP positive embryos were selected for phenotypic readouts. Images were collected using Leica M165 FC stereomicroscopes (Leica Biosystems, Wetzlar, Germany) and analyzed using ImageJ (U.S. National Institutes of Health, Bethesda, MD, USA). To determine epiboly defects, the ratio of the major (x) and minor (y) axis was determined in 11 hpf embryos. Lengths of 3 and 5 dpf anesthetized (0.1% MS-222) embryos were determined by lateral imaging. In situ hybridization was performed as described using myl7-specific probe in PTU-treated 3 dpf embryos (22). Alcian blue staining of the cartilage in PTU-treated 4 dpf embryos was performed as described (48). The ratio between the width of the head (x) and the distance from the back of the head to Meckel’s cartilage (y) was determined.

Statistical analysis was performed using GraphPad Prism 7.05 (GraphPad Software Inc., San Diego, CA, USA) using the analysis of variance complemented by Tukey’s honest significant difference test.

Conflict of Interest statement. The authors declare that they do not have any conflict of interest.