Fig 2: MAGEA proteins are incorporated into EVs.(A) Schematic representation of the purification scheme of EVs. (B) Western blot analysis of COP5 cells and EVs purified from COP5 cells ectopically expressing recombinant MAGEA proteins. The presence of proteins was analysed by specific antibodies against MAGEA4, MAGEA10, TSG101 (T5701; Sigma-Aldrich) and alpha-tubulin (T5168; Sigma-Aldrich). 5% of each EV fraction was loaded to the gel. (C) Western blot analysis of EVs purified from COP5 cells ectopically expressing recombinant MAGEA proteins using anti-E2Tag antibody (for MAGEA4 and MAGEA10) and anti-TSG101 antibody (T5701; Sigma-Aldrich). 5% of each EV fraction was loaded to the gel. (D) Western blot analysis of EVs obtained with ultracentrifugation through stepwise sucrose density gradient (20%, 35%, 45%, 60%) at 120 000 g for 1.5 hours at 4° C. Gradient was divided into 8 fractions and the presence of MAGEA4 protein in each fraction was analyzed with MAGEA4 specific antibody. Top and bottom on the right size of the image depicts the loading place of samples. (E) Physical characterization of EVs as assessed by DLS (Dynamic Light Scattering). The hydrodynamic diameter (Z-average) and polydispersity index (PdI) are shown on the right. (F) Physical characterization of 120K EVs as assessed by NTA (Nanoparticle tracking analysis).

Fig 3: MAGEA2 interacts with the p53 pathway in breast cancer lines. (a) Whole-cell extract (WCE) lysates from WT and TR T47D cells were immunoprecipitated (IP) for either p53 (left panels) or MAGEA2 (middle panels), and subsequent western blots (WBs) were probed for the same proteins, as indicated. Separate WBs (loaded with 5% of the protein levels used for the IP experiments; right panels) were probed for the indicated proteins. (b) H1299 (p53-null) cells were transfected with the pG1PyLuc reporter plasmid (150 ng), a p53 expression vector (10 ng) and increasing amounts of MAGEA2 or MAGEA3 expression vectors, as indicated. (c) As in (b) but with MAGEA2 and/or MAGEA3 expression vectors (100 ng), as indicated. (d and e). As in (c) but with the p21cip-Luc reporter plasmid (150 ng) without the addition of p53 expression plasmid using p53-null H1299 cells (d) or p53 WT HepG2 cells (e). (b–e) Cells were harvested after 48 h. Luciferase values were corrected for transfection efficiency and expressed as fold activity compared with the reporter alone control (set at 1) using data from three independent experiments, each performed in triplicate; error bars indicated the s.e. Student's t-test was used to compare data from the indicated samples, *P<0.05; **P<0.01.

Fig 4: MAGEA2 interacts with the ER. (a) MCF-7-derived MAGEA2-expressing (C18 and C24) and VA lines were incubated with and without 10-7 M tamoxifen (TAM) for 8 days. Nuclear (N) and cytoplasmic (C) extracts (20 µg) were analyzed by western blotting for the indicated antigens. Blotting for lamin A/C and actin confirmed extract integrity. (b) MCF-7VA and C18/C24 cells were transiently transfected with an ERE-luciferase reporter construct (100 ng) and left untreated or given 10-8 M E2 and/or 10-6 M TAM, as indicated, 24 h later. Luciferase values were corrected for transfection efficiency and expressed as fold activity compared with untreated cells (set at 1) using data from three independent experiments, each performed in triplicate; error bars indicate the s.e. Student's t-test was used to compare data from the indicated samples, *P<0.05; **P<0.01. (c) WCE lysates from MCF-7VA and C18/C24 cells treated as in (a) were immunoprecipitated (IP) for ER (middle panels) or MAGEA2 (right panels), and then blots were probed for ER (top row) or MAGEA2 (middle row), as indicated. Control blots (left panels) carrying 5% of the protein used for the IP were additionally probed for Hsc70 as a loading control.

Fig 5: Histone modifications at the MAGEA12 locus in four breast cancer cell lines that have high or low MAGEA12 and MAGEA3 expression. (a) IGV genome browser snapshot showing the ChIP-seq data for H3K4me3, H3K27ac, and H3K79me2 in the MAGEA12 high-expressing (MDAMB468; TNBC and SKBR3; HER2+) and low-expressing (MDAMB231; TNBC and MCF7; Luminal) cell lines. (b) Snapshot of RNA-seq reads at the MAGEA12-associated loci in the four cell lines. (c) qRT-PCR expression of MAGEA12 and MAGEA3 in the four cell lines. (d) Western blot analysis of MAGE-A proteins in the four cell lines.