Animal handling

All experiments were performed according to the guidelines of the Swiss Animal Welfare Law and in compliance with the regulations of the Cantonal Veterinary Office, Zurich (License for animal experimentation ZH151/2014; ZH146/17; ZH203/2020). The animal facility provided standardized housing conditions, with a mean room temperature of 21 °C ± 1 °C, relative humidity of 50% ± 5%, and 15 complete changes of filtered air per hour (HEPA H 14filter); air pressure was controlled at 50 Pa. The light/dark cycle in the animal rooms was set to a 12 h/12 h cycle (lights on at 07:00, lights off at 19:00) with artificial light of approximately 40 Lux in the cage. The animals had unrestricted access to sterilized drinking water, and ad libitum access to a pelleted and extruded mouse diet in the food hopper (Kliba No. 3436; Provimi Kliba/Granovit AG, Kaiseraugst, Switzerland). Mice were housed in a barrier-protected specific pathogen-free unit and were kept in groups of maximum 5 adult mice per cage in standard IVC cages (Allentown Mouse 500; 194 mm × 181 mm × 398 mm, floor area 500 cm2; Allentown, New Jersey, USA) with autoclaved dust-free poplar bedding (JRS GmbH + Co KG, Rosenberg, Germany). A standard cardboard house (Ketchum Manufacturing, Brockville, Canada) served as a shelter, and tissue papers were provided as nesting material. Additionally, crinklets (SAFE® crinklets natural, JRS GmbH + Co KG, Rosenberg, Germany) were provided as enrichment and further nesting material. The specific pathogen-free status of the animals was monitored frequently and confirmed according to FELASA guidelines by a sentinel program. The mice were free of all viral, bacterial, and parasitic pathogens listed in FELASA recommendations.47Nogo-A KO (Nogo-A -/-, B6-RTN4A<tm1/Schwab>30) and Nogo-A fl/fl mice (C57BL/6-Rtn4tm2175Arte48 were generated by the Schwab lab. K14cre;Nogo-A fl/fl mice were obtained by crossing Nogo-A fl/fl (C57BL/6-Rtn4tm2175Arte) mice with K14cre (Tg(KRT14-cre)1Amc; MGI: 2445832) mice. Mouse pups younger than postnatal day 10 (PN10) were sacrificed by decapitation. PN20 and older mice were anesthetized with Ketamine/Xylazine and sacrificed by intracardiac perfusion with phosphate-buffered saline (PBS) followed by Paraformaldehyde (PFA) 4% in PBS. Mouse heads were then separated and post-fixed in PFA 4% overnight at 4 °C. The specimens were further washed with PBS and processed according to the application.

Immunohistochemistry, immunofluorescent staining, in situ hybridisation

5 μm Paraffin sections were rehydrated by incubation in Xylol followed by a series of Ethanol solutions (100% to 30%) and distilled H2O. For immunohistochemistry only, endogenous Peroxidase was quenched by incubation for 30 min, at 4 °C, in 0.3% H2O2 diluted in Methanol. Thereafter, specimens were blocked with PBS supplemented with 2% Fetal Bovine Serum (FBS) and incubated with primary antibodies for 1 h at room temperature. The following primary antibodies were used: human anti-Nogo-A (dilution 1:300),1 rabbit anti-S1PR2 (dilution 1:100; 4385-AP01311PU-N; OriGene Technologies, Rockville, MD, USA), goat anti-NgR1 (dilution 1:100; AF1440, R&D Systems, Minneapolis, MI, USA), rabbit anti-P75/NGFR (dilution 1:200; N3908, Sigma Aldrich, Buchs, Switzerland). The sections were then incubated either with Fluorochrome-conjugated secondary antibodies or with biotinylated secondary antibodies for 1 h at room temperature. The following secondary antibodies were used: biotinylated anti-human (dilution 1:1 000; 709-065-149, Jackson ImmunoResearch, Switzerland), Alexa 568-conjugated anti-rabbit (dilution 1:500) Alexa-647 conjugated anti-mouse (dilution 1:500), Alexa-568-conjugated anti-goat (dilution 1:500) (ThermoFisher Scientific, Basel, Switzerland). For immunohistochemistry, sections were incubated in ABC reagent (Vector Labs, Burlingame, USA) and developed with the AEC detection kit (Vector Labs, Burlingame, USA). Slides were counterstained with Toluidine blue and mounted with Glycergel (DAKO/Agilent, Santa Clara, CA, USA). For immunofluorescent nuclear staining, 4’,6-Diamidino-2-Phenylindole (DAPI; D1306, Thermo Fisher Scientific, Reinach, Switzerland) was used. After immunofluorescent staining, samples were mounted in ProLong™ Diamond Antifade Mountant (P36965, Thermo Fisher Scientific, Reinach, Switzerland), and imaged with a Leica SP8 Inverted Confocal Laser Scanning Microscope (Leica Microsystems- Schweiz AG, Heerbrugg, Switzerland).

Anti-Nogo-A riboprobes were used for in situ hybridization on 14 μm cryosections, as previously described.49 The hybridization signal was detected using the NBT/BCIP substrate solution, sections were mounted with Glicergel® mounting medium (Dako) and imaged with Leica DM6000 microscope equipped with the Leica DFC420C camera. Images were processed with the Leica Application Suite (LAS) software.

Scanning electron microscopy

Fully mineralized lower hemi-jaws were dissected from perfused adult (two months old) wild-type (n = 6), Nogo-A KO (n = 6) and K14cre; Nogo-A fl/fl (n = 3) mice. Soft tissues were removed manually and by incubating the samples in 3% H2O2 overnight. The mandibles were then dehydrated and embedded in Technovit 7200 VLC (Heraeus Kulzer, Wehrheim, Germany). Light-polymerized blocks were mounted on Aluminium stubs, polished and coated with a 10–15 nm thick layer of Carbon, and finally examined using a Tescan EGATS5316 XMSEM (Tescan, Brno, Czech Republic) operated in BSE mode. Elemental composition of enamel was analysed with the aid of energy-dispersive X-ray spectroscopy (EDS). A Si(Li) detector (Oxford Instruments, Wiesbaden, Germany) served for recording EDS spectra using an accelerating voltage of 7 kV, a working distance of 23 mm, and a counting time of 100 s. For the quantitative analysis of these spectra, the Inca energy software (Oxford Instruments) was used. Comparisons between EDS of wild type and Nogo-A KO lower incisors enamel were performed on n = 4 animals per condition; abundance of each element in the two conditions was compared via two-tailed t-test (Graph Pad Prism 8.0).

Transmission electron microscopy (TEM)

Lower hemi-jaws were dissected from perfused adult (approx. 3 months of age) wild type (n = 3) and Nogo-A KO (n = 3) mice and decalcified in 10% EDTA (pH 7.4) for 10 weeks. The samples were then postfixed in 1.33% Os-tetraoxide in 0.067 M s-collidine buffer for 2 h at room temperature. Thereafter, they were dehydrated in ethanol, transferred to propylene oxide and embedded in Epon 812 (Fluka, Buchs, Switzerland). From the resin blocks, thin sections of 80–100 nm in thickness were cut using a Reichert Ultracut ultramicrotome (Leica Microsystems, Heerbrugg, Switzerland) and diamond knives (Diatome, Biel, Switzerland). Sections were collected on copper grids, contrasted with U-acetate and Pb-citrate, and examined in a Philips EM400 T TEM (FEI, Eindhoven, Netherlands) at 60 kV. Micrographs were recorded using a Hamamatsu ORCA-HR camera (Hamamatsu Photonics, Hamamatsu, Japan) and the AMT image acquisition software (Deben, Bury St. Edmunds, UK).

RNA sequencing

Samples were isolated from lower incisors freshly dissected from new-born (PN0) wild-type and K14cre;Nogo-A fl/fl pups. The dental epithelium was isolated by incubating the incisors in Dispase (2 mg/ml in HBSS; D4818, Sigma Aldrich, Buchs, Switzerland). RNA was isolated using the RNeasy Plus Mini Kit (Qiagen AG, Switzerland) and subsequently purified by Ethanol precipitation. The quality of the isolated RNA was determined with a Qubit® (1.0) Fluorometer (Life Technologies, California, USA) and a Bioanalyzer 2100 (Agilent, Waldbronn, Germany). Only those samples with a 260 nm/280 nm ratio between 1.8–2.1, 28S/18S ratio within 1.5-2, and a RIN (RNA integrity number) of >7 were further processed. The TruSeq RNA Sample Prep Kit v2 (Illumina, Inc, California, USA) was used in the succeeding steps. Briefly, total RNA samples (100–1 000 ng) were Poly A enriched and then reverse-transcribed into double-stranded cDNA. The cDNA samples were fragmented, end-repaired and polyadenylated before ligation of TruSeq adapters containing the index for multiplexing Fragments containing TruSeq adapters on both ends were selectively enriched with PCR. The quality and quantity of the enriched libraries were validated using Qubit® (1.0) Fluorometer and the Caliper GX LabChip® GX (Caliper Life Sciences, Inc., USA). The product is a smear with an average fragment size of approximately 260 bp. The libraries were normalized to 10 nmol/L in Tris-Cl 10 mmol/L, pH8.5 with 0.1% Tween 20. The TruSeq PE Cluster Kit HS4000 or TruSeq SR Cluster Kit HS4000 (Illumina, Inc, California, USA) was used for cluster generation using 10 pmol/L of pooled normalized libraries on the cBOT. Sequencing was performed on the Illumina HiSeq 4000 single end 125 bp using the TruSeq SBS Kit HS4000 (Illumina, Inc, California, USA). Individual library size ranged from 30 million to 50 million reads. RNA sequencing analysis was performed using the SUSHI framework,50 which encompassed the following steps: read quality was inspected using FastQC, and sequencing adaptors removed using fastp51; Alignment of the RNA-Seq reads using the STAR aligner52 and with the Ensembl Mus musculus genome build GRCm38 (patch 5, Release 91) as the ref. 53; the counting of gene-level expression values using the ‘featureCounts’ function of the R package Rsubread54; differential expression using the generalised linear model as implemented by the edgeR Bioconductor R package and normalized with the edgeR trimmed mean of M-values (TMM)55 and; Gene Ontology (GO) term pathway analysis using both the hypergeometric over-representation test via the ‘enricher’ function, and gene-set enrichment analysis via the ‘GSEA’ function, of the clusterProfiler Bioconductor R package.56 Differential abundance of splicing isoforms was quantified with sqSeq.57 All R functions were executed on R version 3.5 (R Core Team, 2020) and Bioconductor version 3.7. A gene is marked as DE if it possesses the following characteristics: i) at least 10 counts in at least half of the samples in one group; ii) P < = 0.05; iii) fold change ≥ 0.5. Finally, gene sets were used to interrogate the GO Biological Processes database for an exploratory functional analysis. Contingency tables were constructed based on the number of significant and non-significant genes in the categories and we reported statistical significance using Fisher’s exact test. RNA-sequencing data are available at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE160829.

Immunoprecipitation and mass spectrometry for interactome characterization

Dissected dental epithelia were minced in cold PBS and treated with a hypotonic lysis buffer (20 mmol/L tris-HCl, 75 mmol/L NaCl, 1.5 mmol/L MgCl2, 1 mmol/L EGTA, 0.5% NP-40, and 5% Glycerol). The obtained protein extracts were incubated with 1 mg of 11C7 anti-Nogo-A mouse monoclonal antibody1 and protein A-conjugated Sepharose beads (GE Healthcare); they were then diluted in lysis buffer to a final volume of 1 mL. After 4 h of incubation at 4 °C on a rotating wheel, the beads were spun down and washed three times in lysis buffer. All steps were performed on ice, and all buffers were supplemented with fresh Protease inhibitors (cOmplete, Roche) and 1 mmol/L Phenylmethylsulfonyl Fluoride. The samples were then analysed by liquid chromatography-MS/MS analysis. For liquid chromatography–MS/MS analysis, the protein samples, already dissolved in Laemmli buffer, were submitted to a filter-aided sample preparation (FASP)58 and digested with Trypsin in 100 mmol/L Triethylammonium Bicarbonate buffer overnight. Desalted samples were dried completely in a vacuum centrifuge and reconstituted with 50 mL of 3% Acetonitrile and 0.1% Formic Acid. Each peptide solution (4 mL) was analysed on both Q Exactive and Fusion mass spectrometers (Thermo Scientific) coupled to EASY-nLC 1000 (Thermo Scientific). On the Q Exactive, full-scan MS spectra were acquired in profile mode from 300 to 1 700 mass/charge ratio (m/z) with an automatic gain control target of 3 Å~ 106, an Orbitrap resolution of 70 000 (at 200 m/z), and a maximum injection time of 120 ms. The 12 most intense multiply charged (z = +2 to +8) precursor ions from each full scan were selected for higher-energy collisional dissociation fragmentation with a normalized collision energy of 30 arbitrary unit. Generated fragment ions were scanned with an Orbitrap resolution of 35 000 (at 200 m/z), an automatic gain control value of 5 Å~ 104, and a maximum injection time of 120 ms. The isolation window for precursor ions was set to 2.0 m/z, and the underfill ratio was at 1% (referring to an intensity of 4.2 Å~ 103). Each fragmented precursor ion was set onto the dynamic exclusion list for 90 s. For the Orbitrap Fusion, the “Universal Method” as described in59 was applied. Peptide separation on both instruments was achieved by reversed-phase high-performance liquid chromatography on an in-house packed C18 column (150 mm Å~75 mm, 1.9 mm, C-18 AQ, 120 Å; Dr. Maisch GmbH). Samples were loaded with maximum speed at a pressure restriction of 400 bar and separated with a linear gradient from 3 to 25% solvent B (0.1% Formic Acid in Acetonitrile, Biosolve BV) in solvent A (0.1% Formic Acid in H2O, Biosolve BV) at a flow rate of 250 nL/min. The column was washed after the separation by flushing with 95% solvent B for 10 min, automatically equilibrated before the next injection, and exported to mgf (Mascot generic format) format for subsequent database search. Peak lists were extracted from the instrument raw files using Proteome Discoverer (version 1.4) in combination with the FCC [Functional Genomics Centre of Zurich (FGCZ) Converter Control]60 and exported to mgf format for subsequent database search. All MS/MS samples were analysed using Mascot software (Matrix Science, version 2.4.1). The parameters were set up to search the fgcz_10090_20140715 database (https://fgcz-proteomics.uzh.ch/fasta/fgcz_10090_20140715.fasta, 51 806 entries), with Trypsin indicated as the digestion enzyme, and the fgcz_10090_d_20140715 database (https://fgcz-proteomics.uzh.ch/fasta/fgcz_10090_d_20140715.fasta, 103 351 entries), also assuming Trypsin. Mascot was searched with a fragment ion mass tolerance of 0.050 Da and a parent ion tolerance of 10.0 parts per million. Carbamidomethyl of Cysteine was specified in Mascot as a fixed modification. Oxidation of Methionine was specified in Mascot as a variable modification. Scaffold (version Scaffold_4.4.1, Proteome Software Inc.) was used to validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95% probability by the Peptide Prophet algorithm61 with Scaffold delta-mass correction. Protein identifications were accepted if they could be established at greater than 95.0% probability and contained at least two identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm.62 Proteins that contained similar peptides and could not be differentiated on the basis of MS/MS analysis alone were grouped to satisfy the principles of parsimony. Proteins sharing significant peptide evidence were grouped into clusters. Gene Ontology63,64 was used to analyse the cellular localisation and the participation in biological processes of Nogo-A interactors.

Nuclear / cytoplasm differential mass spectrometry

Dental epithelia were isolated from the lower incisors of PN6 K14cre; Nogo-A pups and control littermates. Cytoplasmic versus nuclear protein fractionation was performed as previously described.38,65 For each sample, the total provided volume was taken and reduced with 2 mmol/L Tris(2-CarboxyEthyl)Phosphine (TCEP) and alkylated with 15 mmol/L Iodoacetamide at 60 °C for 30 min. The sp3 protein purification, digest, and peptide clean-up was performed using a Kingfisher Flex System (Thermo Fisher Scientific) and Carboxylate-Modified Magnetic Particles (GE Life Sciences).66,67 Beads were conditioned following the manufacturer’s instructions, consisting of 3 washes with water at a concentration of 1 µg/µL. Samples were diluted with an equal volume of 100% Ethanol (50% Ethanol final concentration). The beads, wash solutions and samples were loaded into 96 deep well-plates or micro-plates and transferred to the Kingfisher.

Following steps were carried out on the robot: collection of beads from the last wash, protein binding to beads (14 min), washing of beads in wash solutions 1–3 (80% Ethanol, 3 min each), protein digestion (for 4 h at 37 °C with a trypsin/protein ratio of 1:50 in 50 mmol/L TEAB) and peptide elution from the magnetic beads (water, 6 min). The digest solution and water elution were combined and dried to completeness. Afterwards the peptides were re-solubilized with 20 µL of 3% acetonitrile, 0.1% formic acid and 3 µL of indexed retention time (iRT)-peptides (Biognosys) were spiked in each sample for MS analysis. The peptide concentration was determined using a Lunatic (Unchained Labs) instrument. Mass spectrometry analysis was performed on an Orbitrap Fusion Lumos (Thermo Scientific) equipped with a Digital Picoview source (New Objective) and coupled to a M-Class UPLC (Waters). Solvent composition of the two channels was 0.1% Formic Acid for channel A and 0.1% Formic Acid, 99.9% Acetonitrile for channel B. For each sample 0.1 abs of peptides were loaded on a commercial MZ Symmetry C18 Trap Column (100 Å, 5 µm, 180 µm x 20 mm, Waters) followed by nanoEase MZ C18 HSS T3 Column (100 Å, 1.8 µm, 75 µm x 250 mm, Waters). The peptides were eluted at a flow rate of 300 nL/min. After an initial hold at 5% B for 3 min, a gradient from 5 to 24% B in 80 min and 36% B in 10 min was applied. The column was washed with 95% B for 10 min, and afterward, the column was re-equilibrated to starting conditions for an additional 10 min. Samples were acquired in a randomized order. The mass spectrometer was operated in data-dependent mode (DDA), acquiring full-scan MS spectra (300 − 1 500 m/z) at a resolution of 120 000 at 200 m/z after accumulation to a target value of 500 000. Data-dependent MS/MS were recorded in the linear ion trap using quadrupole isolation with a window of 0.8 Da and HCD fragmentation with 35% fragmentation energy. The ion trap was operated in rapid scan mode with a target value of 10 000 and a maximum injection time of 50 ms. Only precursors with intensity above 5 000 were selected for MS/MS, and the maximum cycle time was set to 3 s. Charge state screening was enabled. Singly, unassigned, and charge states higher than seven were rejected. Precursor masses previously selected for MS/MS measurement were excluded from further selection for 20 s, and the exclusion window was set at 10 ppm. The samples were acquired using internal lock mass calibration on m/z 371.1012 and 445.1200. The mass spectrometry proteomics data were handled using the local laboratory information management system (LIMS).68 The acquired raw MS data were processed by MaxQuant (version 1.6.2.3), followed by protein identification using the integrated Andromeda search engine.69 Spectra were searched against a mouse reference proteome (version from 2019-07-09), concatenated to its decoyed fasta database and common protein contaminants. Carbamidomethylation of Cysteine was set as fixed modification, while Methionine oxidation and N-terminal protein acetylation were set as variable. Enzyme specificity was set to Trypsin/P, allowing a minimal peptide length of 7 amino acids and a maximum of two missed cleavages. MaxQuant Orbitrap default search settings were used. The maximum false discovery rate (FDR) was set to 0.01 for peptides and 0.05 for proteins. Label-free quantification was enabled, and a 2 min window for match between runs was applied. In the MaxQuant experimental design template, each file is kept separate in the experimental design to obtain individual quantitative values. Protein fold changes were computed based on Intensity values reported in the proteinGroups.txt file. A set of functions implemented in the R package SRMService (https://rdrr.io/github/protViz/SRMService/) was used to filter for proteins with two or more peptides. A modified robust z-score transformation was applied to remove differences among the sample. Finally, p-values were computed using the t-test with empirical Bayes variance shrinkage.70 If all protein measurements are missing in one of the conditions, a pseudo fold change was computed, replacing the missing group average by the mean of 10% smallest protein intensities in that condition. In addition, we performed gene set enrichment analysis using the R/Bioconductor package fgsea (https://doi.org/10.18129/B9.bioc.fgsea) and used gene sets specified in the molecular signature database (http://www.gsea-msigdb.org/). To apply GSEA to proteomics data, we mapped the UniProt identifiers to Entrez Id’s using the UniProt mapping service. Then, we ordered the protein lists using the log2FC. For cases where several UniProt Id’s were mapped to a single Entrez Id, we averaged the log2FC. All MS data have been deposited in the PRIDE (Proteomics Identifications) Archive database,71 with accession number PXD030386.

Identification of potential Hsf1 binding sites

Potential binding sites were identified using the FIMO motif scanning package.72 As input, we selected the sequences of the Amelx, Ambn, Enam, and Klk4 mouse genes, plus 1 000 base pairs upstream of the TSS and downstream of the annotated loci to include the promoter region and potential proximal downstream regulatory sequences. Hsf1 motifs were obtained from Motifmap.73 Output files are provided within the “Supplementary_File2_FIMO_HSF1motif_search” supplementary file.

LS8 cell culture and analysis

LS8 cells35 were cultured in DMEM (31966047, ThermoFisher Scientific, Basel, Switzerland) supplemented with 10% FBS and 1x Penicillin/Streptomycin (P/S; 15140122, Thermo Fisher Scientific, Basel, Switzerland). For antibody-mediated inhibition of Nogo-A, LS8 cells were treated with 10 μg/mL 11C7 anti-Nogo-A antibody (n = 3),1 while control cells were treated with 10 μg/mL mIgG1 (n = 3). For shRNA-mediated Nogo-A knockdown experiments, LS8 cells were transfected with plasmids for shRNA against Nogo-A and scrambled shRNA control Sigma-Aldrich;SHCLNG, NM_194054 / TRCN0000071689; n = 3 shRNA anti-Nogo-A; n = 3 scrambled shRNA). Transfections were performed with the JetOPTIMUS® DNA Transfection Reagent (Polyplus Transfection, Illkirch, France). For real time PCR analysis, cells were lysed with the lysis buffer from the RNeasy Mini Plus Kit and processed according to the manufacturer instructions. Reverse transcription of the isolated RNA was performed using the iScript™ cDNA synthesis Kit and according to the instructions given (Bio-Rad Laboratories AG, Cressier FR, Switzerland). Briefly, 1 000 ng of RNA were used for reverse transcription into cDNA. Nuclease-free water was added to add up to a total of 15 μL. 4 μL of 5x iScript reaction mix and 1 μL of iScript reverse transcriptase were added per sample in order to obtain a total volume of 20 μL. The reaction mix was then incubated for 5 min at 25 °C, for 30 min at 42 °C and for 5 min at 85 °C using a Biometra TPersonal Thermocycler (Biometra AG, Göttingen, Germany). The 3-step quantitative real-time PCRs were performed using an Eco Real-Time PCR System (Illumina Inc., San Diego CA, USA). 5 μL of SYBR® Green PCR Master Mix reverse and forward primers (200 nmol/L), and 2 ng of template cDNA were added to each well. The thermocycling conditions were: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 55 °C for 30 s and 60 °C for 1 min. Melt curve analysis was performed at 95 °C for 15 s, 55 °C for 15 s and 95 °C for 15 s. Expression levels were calculated by the comparative ΔΔCt method (2−ΔΔCt formula), after being normalized to the Ct-value of the housekeeping gene. Analysis: one-way ANOVA, non-parametric, Tukey correction for multiple comparisons (Graph Pad Prism 8.0).

For imaging analysis, at the end of the culture period, cells were fixed with 4% PFA for 10 min, washed in PBS supplemented with 2% BSA for 30 min and then processed for immunofluorescent staining. Cells were incubated with primary antibodies for 1 h at room temperature. The following primary antibodies were used: mouse anti-Nogo-A.1 (dilution 1:200), rabbit anti-Hsp90 (dilution 1:200; 4877 T, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-S1PR2 (dilution 1:100;4385-AP01311PU-N; OriGene Technologies, Rockville, MD, USA), rabbit anti-HSF1 (dilution 1:100, ab2923, Abcam - Lucerna-Chem AG, Luzern, Switzerland), goat anti-NgR1 (dilution 1:100; AF1440, R&D Systems, Minneapolis, MI, USA), rabbit anti-P75NGFR (dilution 1:200; N3908, Sigma Aldrich, Buchs, Switzerland). Cells were then washed with PBS and incubated with secondary antibodies for 1 hour at room temperature. The following antibodies were used: Alexa 568-conjugated anti-rabbit dilution 1:500), Alexa-647 conjugated anti-mouse (dilution 1:500), Alexa-568-conjugated anti-goat (dilution 1:500) (ThermoFisher Scientific, Basel, Switzerland). Cells were then counterstained with Alexa-488 conjugated phalloidin (A12379, ThermoFisher Scientific, Basel, Switzerland) and DAPI, and imaged with a Leica LS8 confocal laser scanning microscope. For cell surface Nogo-A immunostaining, cells were incubated, without fixation, in 4 °C PBS supplemented with 10 μg/mL 11C7 anti-Nogo-A antibody and incubated for 1 h at 4 °C. Cells were then fixed and processed as described above. Images were analyzed with Fiji / ImageJ.74