The multifunctional role of SPANX-A/D protein subfamily in the promotion of pro-tumoural processes in human melanoma

Human sperm protein associated with the nucleus on the X chromosome (SPANX) genes encode a protein family (SPANX-A, -B, -C and -D), whose expression is limited to the testis and spermatozoa in normal tissues and various tumour cells. SPANX-A/D proteins have been detected in metastatic melanoma cells, but their contribution to cancer development and the underlying molecular mechanisms of skin tumourigenesis remain unknown. Combining functional and proteomic approaches, the present work describes the presence of SPANX-A/D in primary and metastatic human melanoma cells and how it promotes pro-tumoural processes such as cell proliferation, motility and migration. We provide insights into the molecular features of skin tumourigenesis, describing for the first time a multifunctional role of the SPANX-A/D protein family in nuclear function, energy metabolism and cell survival, considered key hallmarks of cancer. A better comprehension of the SPANX-A/D protein subfamily and its molecular mechanisms will help to describe new aspects of tumour cell biology and develop new therapeutic targets and tumour-directed pharmacological drugs for skin tumours.


Results
The SPANX-A/D protein subfamily is expressed in human melanoma cells. SPANX-A/D proteins belong to the so-called CTAs that are physiologically expressed in normal male germ cells and aberrantly expressed in various cancers 6,13 . Immunofluorescence analyses were performed to evaluate the presence of the SPANX-A/D protein subfamily in several cancer cell lines. The SPANX-A/D subfamily was highly expressed in A375, MelHO and Colo-800 melanoma cell lines (Fig. 1A), as well as in several cell lines derived from distinct tumours, such as SW480 and HCT-8 (colorectal adenocarcinoma), HeLa (epithelioid cervix carcinoma), A2780 (ovary adenocarcinoma), SMS-KCNR (neuroblastoma cell line), and MCF-7 (mammary adenocarcinoma) ( Supplementary Fig. S1). Remarkably, in all the tested cell lines, SPANX-A/D immunolabelling was prominently nuclear, with a faint cytoplasmic signal noted in some cases (Fig. 1A). As we expected, no signal was observed in human melanocytes (HEMn-MP), proving the specificity of the primary antibody. In negative control samples, in which incubation with the primary antibody was omitted, fluorescence was not observed.
A more in-depth analysis of the melanoma cell lines by immunoblotting confirmed the presence of SPANX-A/D proteins in both primary (A375 and MelHO) and metastatic (Colo-800) melanoma cell lines (Fig. 1B). The anti-SPANX polyclonal antibody labelled an 11-kDa band. No immunoreactivity was observed when the primary antibody was omitted (data not shown). To further characterise the expression of the SPANX-A/D subfamily in melanoma cells, we sought to determine the presence of different protein isoforms in A375 cells. We detected a single peptide (TSESSTILVVR) that corresponds to a nuclear localisation signal (NLS) shared by all SPANX-A, -B, -C and -D isoforms (Fig. 1C). Hence, proteomic analysis corroborated the presence of SPANX-A/D in A375 cells but could not determine whether one, two, three or all members were co-expressed. According to our results, SPANX-A/D can be phosphorylated in human spermatozoa 7 . To uncover whether this subfamily is also phosphorylated in A375, we conducted proteomic studies. Notably, no phosphorylated SPANX-A/D residues were found in this analysis, suggesting that these proteins are mostly in a non-phosphorylated form in A375 cells.

SPANX-A/D expression promotes the proliferation, motility and migration of human melanoma cells.
Recently, SPANX-A/D proteins have emerged as strong candidates for cancer immunotherapy 14,15 .
However, the role of this family in skin tumourigenesis remains largely unknown. SPANX-A/D proteins have been detected in metastatic melanoma cells 9 , but their contribution to metastatic development and the potential molecular underlying mechanisms have not yet been determined. To further investigate the potential significance of SPANX-A/D in the proliferation and migration capacity of melanoma cells, we used lentiviral-mediated delivery of short hairpin (sh) RNAs to generate stable knockdown variants of the A375 human melanoma cell line with reduced expression of SPANX-A/D (A375 SPANX-KD ) and a control cell line (A375 CTRL ). We generated and checked three different shRNAs, and the most effective shRNA was used for further experiments (data not shown). The depletion of SPANX-A/D was confirmed at the mRNA and protein levels by qRT-PCR and immunoblotting, respectively ( Fig. 2A,B). A reduction of approximately 65% in SPANX-A/D mRNA and protein levels was observed in cells transfected with the most effective shRNA compared with scramble shRNA-transfected cells. Furthermore, the SPANX-A/D immunofluorescence signal was absent from the nucleus of A375 SPANX-KD cells (Fig. 2C).
We first analysed tumour cell proliferation using the PrestoBlue viability assay. Reduced expression of SPANX-A/D resulted in the decreased cell viability of A375 cells. A375 SPANX-KD cells exhibited 35% reduced proliferation following 24 h of incubation compared with A375 CTRL cells (Fig. 2D). Additionally, we analysed the ability of SPANX-A/D to modulate A375 tumour cell motility. Wound healing assays revealed reduced motility of SPANX-A/D knockdown cells (Fig. 2E). A375 CTRL cells closed 45% of the wound, while A375 SPANX-KD cells only closed 35% of the scratch. Therefore, SPANX-A/D deficiency led to a 10% reduction in wound closure. Finally, we conducted Transwell assays to evaluate the Transwell migration capacity of SPANX-A/D knockdown cells. An average of 140 A375 CTRL cells per 20 × field migrated through an 8-µm pore filter, whereas an average of only 90 A375 SPANX-KD migrated through the filter (Fig. 2F). Therefore, according to our results, the Transwell migration capacity of SPANX-A/D-deficient melanoma cells was reduced by approximately 35% relative to that of control cells. Taken together, our results show that the depletion of SPANX-A/D compromises the capacity of A375 melanoma cells to proliferate and migrate.

SPANX-A interacts with proteins involved in crucial nuclear processes in melanoma cells.
To gain insight into the molecular mechanisms by which SPANX-A/D might promote proliferation and migration in melanoma cells, we aimed to study the interactome of SPANX-A. Briefly, YFP-SPANX-A was expressed in A375 cells and pulled down in quadruplicate using GFP-Trap. All the precipitated proteins were analysed by mass spectrometry and subjected to label-free quantitative proteomics. We detected 246 potential interactors of SPANX-A (Supplementary Table S1). To investigate the biological and molecular functions in which these   www.nature.com/scientificreports/ putative SPANX-A interactors might be involved, we performed two different gene ontology analyses using the PANTHER functional annotation tool. SPANX-A seems to participate in various biological processes, including the regulation of mRNA processing and stabilisation, cell metabolism and the transport of proteins and RNA to Cajal bodies and other cell compartments (Fig. 3A). Regarding molecular function, the most enriched processes were those related to RNA transcription, mRNA binding and alternative splicing, and protein translation and folding. Taken together, these results highlight the possible involvement of SPANX-A in crucial nuclear processes.
To obtain more detailed idea about the nature and roles in which putative SPANX-A proteins are involved, we checked them in the UniProt database (Supplementary Table S2). Based on the data collected in the curated database, all the proteins that constitute our list of putative SPANX-A binding proteins were classified into 14 categories (Fig. 3B). Consistent with the results obtained in the gene ontology analysis, most of the proteins are involved in key nuclear functions, such as chromatin organisation, RNA transcription and protein translation. Notably, our list of putative SPANX-A proteins includes 15 ribonucleoproteins and 64 ribosomal proteins. www.nature.com/scientificreports/ Additionally, of the 246 putative SPANX-A interactors, 19, 17 and 14 proteins were involved in cell metabolism, cell structure and cell survival, respectively. Next, we validated the SPANX-A interaction with proteins related to nuclear functions, such as Lamin-A/C (LMNA), one of the proteins that showed the highest MS intensity. Co-immunoprecipitation analyses confirmed the physical interaction between both proteins. LMNA was detected in YFP-SPANX-A-containing immune complexes (Fig. 3C) and co-localised with SPANX-A over the nuclear envelope (Fig. 3D). Consistent with the results obtained in gene ontology analysis, we also identified a broad collection of proteins related to protein translation and folding, such as ribosomal proteins and chaperones (Fig. 3B). Specifically, immunofluorescence analyses confirmed the co-localisation of YFP-SPANX-A and 60S ribosomal protein L8 (RPL38) in the cytoplasm and heat shock cognate 71 kDa protein (HSPA8) in the nucleus of A375 cells (Fig. 3E).
To provide further information about the molecular mechanisms of SPANX-A/D in the promotion of tumorigenic processes, we evaluated the expression of Lamin A/C and HSPA8 in SPANX knockdowned melanoma cells (A375 SPANX-KD ). As can be observed in Fig. 3F, the expression of both Lamin A/C and HSPA8 did not change in A375 SPANX-KD cells, indicating that the expression of both interactors is not dependent to SPANX-A/D expression. Our results, therefore, indicate that the interaction between SPANX-A/D and these proteins is crucial for the promotion of tumourigenic processes in human melanoma, rather than the regulation of their protein expression.

Discussion
Over the last twenty years, many studies have analysed the presence of the SPANX-A/D subfamily in various cancers, such as breast, myeloma, haematological, melanoma, bladder and prostate carcinomas 3,[8][9][10][11][12] , to clarify the function of the protein subfamily in carcinogenesis. Through a combination of functional and proteomic approaches, we proved that SPANX-A/D proteins play a multifunctional role in skin tumourigenesis, providing insight into how this protein family may promote pro-tumoural processes in human primary melanoma cells (Fig. 4).
We confirmed the presence of SPANX-A/D proteins in different cancer types, such as colorectal cancer, cervical-uterine cancer, neuroblastoma, mammary cancer and melanoma cells. Although higher levels of SPANX-A/D are reported to be associated with more aggressive skin tumours 9 , our results show the presence of the SPANX-A/D protein family in both primary and metastatic melanoma cell lines presenting nuclear localisation. However, we have previously reported that the NLS of SPANX-A/D proteins is strongly phosphorylated in human spermatozoa 7 . However, surprisingly, we could not find any phosphorylated SPANX-A/D residues at the NLS in A375 cells. Considering that the SPANX-A/D protein family is mainly localised in the nucleus of melanoma cell lines, our findings confirm that the phosphorylation state of the NLS is not crucial for nuclear SPANX-A/D translocation either in spermatozoa 7 or in A375 cell lines.
SPANX-A/D proteins play a role in the invasion and/or metastasis of several tumours. Our results, together with other studies, describe the presence of SPANX-A/D proteins in metastatic melanoma 9 , and their expression correlates with liver metastasis in colorectal cancer patients 11 . Given the potential significance of SPANX-A/-C/-D in the proliferation and migration 12 of breast cancer cells, we further investigated the function of SPANX-A/D proteins in melanoma cells. The lack of the protein family in the A375 cell line led to reduced proliferation, cell motility and migration, indicating that the SPANX-A/D protein family promotes different pro-tumoural processes, as previously described in breast and lung cancer 12,16 . Uncontrolled cell proliferation and migration represent the essence of neoplastic disease. To elucidate the underlying mechanisms of the pathological role of SPANX-A/D in these pro-tumoural processes, we performed interactome analysis of YFP-SPANX-A overexpression in the A375 melanoma cell line. SPANX-A co-immunoprecipitates with 246 proteins that are involved in certain hallmarks of cancer 17 , such as nuclear functions, metabolism and cell survival. SPANX-A co-precipitates with proteins involved in cell growth and proliferation (such as mitogen-activated protein kinase 1 (MAPK1) and F-box only protein 50 (NCCRP1)), cell adhesion (such as carcinoma-like desmoglein-1 (DSG1) and desmocollin-1 (DSC1), which are involved in different types of cancer [30][31][32][33] and cytoskeletal proteins (such as actin, tubulins and keratins, which are associated with the capacity of cells to migrate and invade new tissues 18 ).
However, the SPANX-A/D protein family may be involved in other uncharacterised biological functions in melanoma cells. According to our proteomic analysis, the SPANX-A/D protein family plays a multifunctional role in melanoma cells, as we have previously reported in human spermatozoa 7 . Considering that this protein family is mainly expressed in the nucleus of the A375 melanoma cell line, SPANX-A interactors that are involved in nuclear functions show the highest intensity values. SPANX-A seems to participate in various nuclear processes, including chromatin organisation, RNA processing and stabilisation, RNA binding, alternative splicing and RNA transcription. Our results show that SPANX-A interacts with LMNA, a finding that is consistent with previous studies performed with SPANX-C 12 , and both proteins co-localise at the nuclear envelope. LMNA is a component of a meshwork of nuclear lamina proteins that underlie the inner nuclear membrane and provide structural stability to the nucleus 19 . LMNA, which is a gene expression regulator, modulates chromatin accessibility and influences the ability of transcription factors to interact with DNA 20 by scaffolding the formation of multiprotein complexes 21 . SPANX-A/D may induce cell proliferation and migration in melanoma cells by forming multiprotein complexes that alter nuclear function because SPANX-A not only interacts with LMNA but also coimmunoprecipitates with several chromatin regulators, such as histones, and a wide range of RNA www.nature.com/scientificreports/ processing proteins involved in RNA stabilisation, translation and alternative splicing, among others. Because aberrant gene expression and abnormal functioning of nuclear processes have been extensively associated with cancer progression in different tumours [22][23][24] , our findings indicate that SPANX-A/D may play a role as a scaffold of multi-protein complexes and recruit proteins related to several nuclear functions to promote pro-tumoural processes in melanoma cells. However, we also identified a broad collection of proteins involved in protein translation, folding and targeting to cell compartments, such as ribosomal proteins or chaperones, as potential SPANX interactors. Specifically, the HSPA8 chaperone and ribosomal protein RPL38 co-localise with SPANX-A in the nucleus and cytoplasm of the A375 cell line, respectively. HSPA8 is usually expressed in the cell nucleoplasm and has been proposed as a biomarker for the early diagnosis of endometrial carcinoma 25 . This chaperone belongs to the spliceosome complex 26 , which is involved in mRNA splicing 27,28 , providing new insights into the role of SPANX A/D in mRNA alternative splicing. Although correct protein folding is essential for the proper functioning of cells, protein transport is not less important. Protein transport regulators are crucial in the mediation of cancer cell biology, encompassing uncontrolled cell growth, invasion and metastasis 29 .
Additionally, SPANX-A also co-precipitates with proteins involved in cell metabolism. The capability to modify or reprogram cellular metabolism to most effectively support neoplastic proliferation is an emerging hallmark of cancer 17 . Otto Warburg described for the first time the anomalous functioning of energy metabolism in cancer cells 30,31 . Cancer cells can reprogram their glucose metabolism, and thus their energy production, by limiting their energy metabolism largely to glycolysis rather than mitochondrial oxidative phosphorylation, leading to a lower efficiency of ATP production even in the presence of oxygen. SPANX-A co-precipitates with specific enzymes that participate in glucose catabolism, such as pyruvate kinase (PKM), D-3-phosphoglycerate dehydrogenase (PHGDH) and pyruvate dehydrogenase E1 component subunit alpha (PDHA1), which have also been identified as oncogenes in malignant tumours not only in melanoma cells but also in colorectal or breast cancer cells 32,33 . Because reprogramming of energy metabolism is linked to cell proliferation in cancer 33 , SPANX-A/D proteins, through their interaction with several metabolic proteins, could be essential in the reprogramming of meeting energy demands for cell proliferation and prompt skin tumourigenesis. Overall, SPANX-A/D proteins www.nature.com/scientificreports/ are emerging as strong candidates for cancer immunotherapy 9,11,12 . In this regard, a more detailed understanding of the pathological role of this protein family in skin tumourigenesis is required to fulfil its therapeutic potential. Our results provide new molecular insight into the pathological role of SPANX-A/D in carcinogenesis. This protein family plays a multifunctional role in human melanoma, promoting pro-tumoural processes by regulating several hallmarks of cancer, such as nuclear functions and organisation, energy metabolism and cell survival. These findings indicate that the SPANX-A/D protein family may have implications beyond immunotherapy and represent previously unrecognised functions for tumour cell biology, which will be crucial to develop new therapeutic targets for skin tumours. Plasmids and transfection. The plasmid encoding YFP-SPANX-A has been previously described 7 . For transfection experiments, A375, MelHO and Colo-800 melanoma cells were seeded onto 12-well or six-well tissue culture plates. For immunohistochemistry experiments, sterile glass coverslips were placed in the wells before cell seeding. Twenty-four hours later, Lipofectamine 2000 reagent (Thermo Fisher Scientific) was used for cell transfection according to the manufacturer's protocol. To assess transfection efficiency, cells were fixed with 3.7% formaldehyde in PBS for 30 min, and then the coverslips were mounted onto slides using Vectashield Antifade Mounting Medium with DAPI (Vector Laboratories, USA). Image analysis with ImageJ (National Institutes of Health; Bethesda, MD, USA) (1.48v) (https ://image j.nih.gov/ij/) software was used to analyse the intensity of YFP fluorescence. RT-qPCR assays. Total RNA was extracted using a PureLink RNA Mini kit (Life Technologies Inc., CA, USA) according to the manufacturer's instructions. RNAse-free DNase I was used to prevent DNA contamination. The RNA concentration and purity were assessed by absorbance at 260 nm using a Synergy HT spectrophotometer (Bio-Tek, Winooski, VT, USA). Reverse transcription (RT) was performed in a 20-µL reaction volume with 1 µg of total RNA using an iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) to synthesise first-strand cDNA, following the manufacturer′s guidelines. Afterwards, RT-qPCR was performed to check the relative expression level of SPANX-A/C/D genes using tubulin as an internal control. Gene-specific amplification was performed using the CFX96 Real-Time System (Bio-Rad). Quantification was performed using cDNA samples from three separate RNA isolations. The reactions were performed in a total volume of 10 µL containing 50 ng of cDNA, 5 µL of SYBR Green master mix (BioRad) and 200 nM of each primer. The following primers were used: SPANX-A/C/D forward: 5′-AAC GAG ATG ATG CCG GAG AC-3′; SPANX-A/C/D reverse: 5′-TTT GGA GGG GGT TGA TTC TG-3′; βIII-Tubulin forward: 5′-CCA GCT GCA AGT CCG AGT -3′; βIII-Tubulin reverse: 5′-CGC CCA GTA TGA GGG AGA T-3′.

Generation of stable SPANX-A/D knockdown (KD) cells. A stable SPANX-
Protein extraction and Western blotting. For the protein expression analyses, cells were lysed using ice-cold RIPA buffer, and the protein concentration was determined as previously explained 7  www.nature.com/scientificreports/ were diluted in Laemmli sample buffer containing dithiothreitol (DTT) and boiled at 96 °C for 5 min. Thirty micrograms of total protein from melanoma cell lines was loaded onto a 12% resolving gel and transferred to polyvinylidene fluoride membranes. The membranes were blocked with Blotto buffer 7 for 1 h and then incubated with a rabbit polyclonal anti-SPANX-Antibody (ab119280; Abcam UK; diluted 1:500 in blocking solution), a rabbit polyclonal anti-Lamin A/C (A0249; Abclonal MA USA; diluted 1:1000), a rabbit polyclonal anti-HSPA8 (A14001; Abclonal; 1:500) or a mouse monoclonal anti-alpha tubulin (T5168; Sigma; diluted 1:4000). After washing in Blotto buffer, the membrane was incubated for 1 h with peroxidase-conjugated secondary antibodies (goat anti-rabbit IgG HRP (sc-2004) and donkey anti-mouse IgG HRP (sc-2314), Santa Cruz Biotechnology, TX, USA; diluted 1:1000). Finally, the membranes were evaluated for peroxidase activity by enhanced chemiluminescence with a self-prepared reagent.

In gel digestion.
To evaluate the expression of the different SPANX isoforms in the A375 melanoma cell line, we followed the protocol described by Urizar-Arenaza et al 7 . Soluble and insoluble protein fractions were loaded onto a precast gradient gel and subjected to reduction, alkylation and digestion using trypsin. Tryptic peptides were extracted from the gel, dried, concentrated and desalted to be separated by liquid chromatography and analysed by tandem mass spectrometry (LC-MS/MS) 7 . Cell viability assay. To compare the proliferation of A375 SPANX-KD and A375 CTRL cell lines, 5 × 10 3 cells were seeded into 96-well plates in complete medium (DMEM supplemented with l-glutamine and 10% FBS) (Sigma-Aldrich) and incubated overnight. After 18 h, the cells were considered to be in T = 0. After further incubation for 24 h, cell viability was measured using PrestoBlue Viability Reagent (Life Technologies, Thermo Scientific, MA, USA) following the manufacturer's instructions. Briefly, the medium was replaced with PrestoBlue diluted 1/10 in complete medium and the viability was measured after 2 h of incubation. The results shown are the means of three different experiments, and the error bars indicate the SEM.

Analysis of SPANX isoforms by LC
Wound healing assay. The involvement of SPANX-A/D in the motility potential of A375 melanoma cells was investigated using the wound healing assay. A375 SPANX-KD or A375 CTRL cells were plated in 24-well plates and incubated for 18 h. Next, the medium was replaced with fresh complete medium supplemented with mytomicin (5 µg/mL) (Sigma-Aldrich) to inhibit cell proliferation and incubated for 90 min. A wound was made in each well by scratching with a 200-µl tip, and the medium was again changed to fresh complete medium after extensive washing of the detached cells. Using an inverted light microscope (Zeiss Axioscope), photographs were taken at T = 0 and after 24 h of incubation (T = 24 h), and the wound area was measured using ImageJ software (1.48v) to establish the initial wound area. The closed wound area was calculated as the wound area at T = 0 minus the area at T = 24. The results shown are the means of three independent replicates, and the error bars indicate the SEM.
Transwell migration assay. www.nature.com/scientificreports/ Co-immunoprecipitation and in-solution digestion. For the SPANX-A interactome, A375 melanoma cells were transfected with the YFP-SPANX-A plasmid as described above. After protein extraction using Co-IP buffer (100 mM NaCl, 20 mM Tris-HCl, 1% NP-40, and complete protease inhibitor cocktail (Complete tablets, Roche)), the pull down was performed in quadruplicate with GFP-Trap_Magnetic Agarose (Chromotek, Germany) following the manufacturer's instructions. The precipitated protein complexes were independently recovered, washed and eluted with guanidinium hydrochloride 8 M pH 8 at 70 °C for 15 min. The eluted proteins were then reduced, alkylated and subjected to in-solution digestion with trypsin (Roche Diagnostics) overnight at 37 °C. The resulting peptides were desalted using C18 Micro SpinColumns (Harvard apparatus), dried in a speed-vac centrifuge (Thermo Scientific) and resuspended in 0.1% FA before LC-MS/MS analysis.
Interactome analyses by LC-MS/MS. Mass spectrometry analyses were conducted using a Q-Exactive HF-X mass spectrometer (Thermo Scientific, Bremen, Germany) connected to an EASY-nanoLC 1200 System (Thermo) using a nanospray flex ion source (Thermo). Desalted peptides were loaded onto an Acclaim Pep-Map100 precolumn (75 μm × 2 cm, Thermo Scientific) connected to an Acclaim PepMap RSLC (75 μm × 25 cm, Thermo Scientific) analytical column 34 . To elute peptides from the column, we used the following gradient: 120 min from 2.4 to 24%, 2 min from 24 to 32% and 12 min at 80% acetonitrile in 0.1% formic acid at a flow rate of 300 nL min −1 . Full MS scans were obtained from m/z 375 to 1800 with a resolution of 60,000 at m/z 200. The 10 most intense ions were fragmented by higher energy C-trap dissociation with a normalised collision energy of 28, and MS/MS spectra were documented with a resolution of 15,000 at m/z 200. The maximum ion injection times were 50 ms and 100 ms, whereas AGC target values were 3 × 10 6 and 1 × 10 5 for survey and MS/MS scans, respectively. To avoid repeat sequencing of peptides, dynamic exclusion was applied for 20 s. Singly charged ions, ions with unassigned charge states and ions with charge states above 5 were also ignored from MS/MS. Data were obtained using Xcalibur software (v.4.0) (Thermo Scientific) 34 .
Data processing and bioinformatics. For the SPANX-A/D isoform analyses, raw files were searched against the combined human database 2015.08 UniProt (with 42,122 sequence entries) and TrEMBL (with 49,496 sequence entries) using the MaxQuant platform version 1.5.2.8 with an Andromeda search engine. To study the SPANX-A/D interactome, raw files were searched against the UniProt-SwissProt human database (version 2018_11, 42424 entries) using the MaxQuant 35 platform (version 1.6.0.16) with its internal search engine Andromeda. The precursor and fragment tolerances were 4.5 and 20 ppm, respectively. A peak list was generated using the Quant element of MaxQuant and the following parameters: a maximum of 2 missed cleavages were allowed, and enzyme specificity was set to trypsin. Additionally, carbamidomethyl (C) was chosen as a fixed modification and variable modifications included oxidation (M), deamidation (NQ) and phospho_STY (STY). The peptide and protein FDR were 0.01, the site FDR was 0.01, the max. peptide PEP was 1, the min. peptide length was 7, and the min. unique peptides and peptides were 1. For protein quantitation in interactome studies, only unmodified peptides were used. The match between runs option was enabled with a 1.5-min match time window and a 20-min alignment window to match identification across samples, and normalised spectral protein label-free quantification (LFQ) intensities were calculated using the MaxLFQ algorithm. According to the protein group assignment performed by MaxQuant, the identified proteins were determined after removing the contaminants, reverse hits and proteins identified only by site. We only considered proteins identified with ≥ 2 peptides and ≥ 1 unique peptide. Additionally, phosphopeptide data were filtered by FDR < 1%, and only the phosphosites displaying a localisation probability above 0.75 were considered confident phosphorylated sites (Class I sites). From all detected proteins, we considered putative SPANX-A interactors that were identified in at least three of the four YFP-SPANX-A pull-downs. The PANTHER (v13.1) functional annotation tool (http:// geneo ntolo gy.org/) was used to detect the overrepresented gene ontology (GO) terms "biological process" and "molecular function" within the possible SPANX-A interactors. www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.