Significant interactants of Delta Np73 alpha in the nuclear/organelle fraction

# Interactome-Delta-Np73alphaInteractome of the oncogenic delta Np73 alpha isoform in human papillomavirus 38 E6/E7-transformed keratinocytes

We have performed affinity purification-mass spectrometry (AP-MS) to characterize the delta Np73 alpha interactome using human keratinocytes immortalized by the beta-HPV38 E6 and E7 oncoproteins as a model of cellular transformation (named 38HK hereafter).  To this end, we generated two stable cell lines, expressing either delta Np73 alpha fused to the N-terminus of the tandem affinity purification (TAP) tag (delta Np73 alpha-TAP) or TAP alone (negative control).To discriminate between cytoplasmic versus nuclear or other organelle interactants, 38HK Np73-TAP and TAP 38HK cells were fractionated. Briefly, 38HK cells were lysed mechanically in hypotonic buffer and centrifuged. The supernatant, corresponding to the cytoplasmic extract, was recovered. The pellet, containing nuclei and other organelles, was resuspended in buffer containing NaCl (250 mM) and glycerol (20%), incubated on ice and centrifuged. The supernatant from this second centrifugation corresponds to the nuclear/organelle extract. Then, extracts were processed by tandem affinity purification. Protein complexes were eluted from calmodulin beads and analysed by SDS-PAGE and Western blotting or silver staining. Protein levels were quantified by the Bradford assay.

Eluates from calmodulin beads were precipitated with trichloroacetic acid, the pellet washed with acetone and dissolved with 2M urea. Samples were digested with 100 ng of trypsin at 37 °C for 10 hours. The resulting peptide mixtures were directly analyzed by MS with a nanoLC U3000 in-line coupled with an Orbitrap Elite mass spectrometer using a nano-electrospray source (Thermo Scientific). MS measurements were performed on one biological sample per condition. Technical variability was estimated from three MS acquisitions for each sample (technical replicates).  Peptides were separated using a C18 column (75µm x 25 cm) with a 35 minutes linear gradient from 5% to 50% buffer B (A: 0.1% trifluoroacetic acid in H2O / B: 80% ACN, 0.08% trifluoroacetic acid in H2O). The mass spectrometer was operated in data-dependent mode with survey scans from m/z 300-1650 acquired in the Orbitrap at a resolution of 120,000 at m/z 400. Proteins were identified using Proteome Discoverer 2.5 software (Thermo Scientific), with false discovery rate (FDR) < 1% and at least one unique peptide. Label free quantification was based on eXtracted Ion Chromatography (XIC) using the Minora node from Proteome Discoverer. Precursor and fragment mass tolerance were set at 7 ppm and 0.5 Da, respectively. Oxidation (M) was set as variable modification, and Carbamidomethylation (C) as fixed modification. The human fasta database with 20419 sequences was obtained from Uniprot (https://www.uniprot.org/proteomes/UP000005640). Statistical analyses were performed using a Perseus 1.6.1.5 according to the processing workflow described by the authors 14 . The repeated t-test used for Volcano plot analyses was based on standard parameters of Perseus (FDR 5%, S0=1).

The Excel file contains the protein candidates (i.e. fold change >2 and FDR < 0.05)