Interaction between MageA2 and p53. (A) Immunoprecipitation (IP) of endogenous p53/Mage protein complex in U2OS cells using anti-p53 polyclonal Ab or preimmune IgG. (B) IP in H1299 cells transfected with HA-MageA2 and the indicated p53 deletions. (C) IP assay similar to that in B but using p53 deletions expressing HA-tagged 94–298 (HA-p53DBD) and HA-p53 298–393 (HA-p53CTD). (D) In vitro binding assay using recombinant/purified GST and GST–p53 fusion protein incubated with IVT ³⁵S-labeled HA–MageA2 (IVT MageA2). (E) In vitro binding assay using recombinant/purified GST and GST–MageA2 fusion protein incubated with IVT ³⁵S-labeled domains of p53:p53NTD (1–98), p53DBD (94–298), and p53CTD (298–393) as indicated. ∗, Ig heavy chain; ∗∗, Ig light chain.

MageA2/HDAC complex deacetylates p53 and histones surrounding p53-binding sites. (A) DNA-damage response in melanoma cells expressing low (15392M) or high (13923M) MAGE-A levels, after addition of 20 μM ET. (B) p53 modifications in 15392M and 13923M melanoma cells after ET damage (20 μM). (C) Similar to B but using 300 nM TSA plus ET (20 μM) treatment. (D) ChIP assay followed by quantitative PCR using real-time PCR. Protein/DNA complexes were immunoprecipitated from 15392M and 13923M melanoma cells before and after ET treatment using the indicated Abs. AcH3, acetylated histone H3 Ab. Data are expressed as the ratio between damaged/undamaged ChIP values, indicating the enrichment of each protein to the indicated promoter after damage.

MageA2 expression represses p53 function. (A) H1299 cells (p53-null) transfected with p53 or in combination with MageA1, -A2, and -A6 together with the p53-responsive promoter pG13LUC. Value corresponding to p53 transfection was reported to 100. (B) p53-specific reporter gene assay as in A using p53 and MageA2 with different p53-responsive promoters. (C) Western blot of M20 cells (HA–MageA2-inducible U2OS cells) expressing (+PonA) or not (No PonA) HA–MageA2. p53, Bax, and p21 protein levels were determined at the indicated time points after treatment with 10 μM ET. (D) Determination of p53, Bax, and p21 protein levels after ET treatment in U2OS cells transfected with control siRNA (siCont) or Mage siRNA (siMage). (E) Apoptosis determination by Annexin V assay coupled to FACS analysis (10,000 counted cells for at least three independent experiments). M20 cells expressing (+PonA) or not (No PonA) HA-MageA2 were treated with 20 μM ET for 36 h. (F) Similar experiment as in E, but performed in U2OS cells previously silenced with siRNA as indicated.

MageA2 recruits HDAC3 to p53. (A) Time-course experiment in M20 cells after addition of 10 μM ET. PonA was added 15 h before ET treatment. DO1 Ab was used for total p53, whereas anti-P-p53Ser15 (P-p53) and anti-Ac-p53Lys382 (Ac-p53) were used for specific phosphorylation and acetylation. (B) Similar to A but using 300 nM TSA plus ET treatment on M20 cells expressing or not expressing HA–MageA2. (C Upper) IP of transiently transfected 293T cells with HA–MageA2. (C Lower) In vitro binding assay using recombinant/purified GST and GST–MageA2 fusion protein incubated with IVT ³⁵S-labeled HDAC3. (D) IP of transiently transfected 293T cells with HA–MageA2 and Flag–HDAC3 expression vectors. (E Upper) ChIP performed in H1299 cells transfected with pBax-Luc, p53, and Flag-tagged construct as indicated. Samples were immunoprecipitated (ChIP) or not (input) by using anti-Flag Ab, followed by pBax–Luc PCR amplification (see diagram in Fig. 7A). (E Lower) Control of the indicated transfections by Western blot. (F Upper) IP of transiently transfected H1299 cells with HA–MageA2 and Flag–HDAC3. Lanes 1 and 2 show the absence and presence of MageA2/HDAC3 complex, respectively. (F Lower) In vitro deacetylation assay of ¹⁴C-acetylated histones (Ac-Hist) by immunoprecipitated complex shown in Lanes 1 (IP-1) and 2 (IP-2) of Upper. Input indicates mock-treated ¹⁴C-acetylated histones. MW, ¹⁴C-labeled protein molecular mass; #, unspecific band; ∗, Ig heavy chain.

MAGE-A expression correlates with resistance to DNA-damage-induced apoptosis. (A) Apoptosis determination by subG1 by FACS in 15392M and 13923M cells. wt-p53 melanoma cells were treated with the indicated concentration of ET for 48 h. Where indicated, 300 nM TSA was added with ET. (B) Similar to A but using melanoma cell lines harboring mutant p53. (C Left) Apoptosis determination by subG1/FACS analysis in 13923M cells cotransduced with siRNA vector, pSR (empty), or pSRM (Mage knockdown), and pBabe (empty) or pBabe-EGFP-TNVp53OD (p53 inactivation) as shown. Cells were treated with 40 μM ET for the indicated time. (C Right) Determination of MAGE-A protein levels and expression of GFP-p53OD in 13923M-pSR and 13923M-pSRM cells using anti-MAGE and anti-GFP polyclonal Ab, respectively. (D) Short-term cell lines obtained from melanoma biopsies harboring wt-p53 were grouped depending on expression level of MAGE-A (see Fig. 7B). Melanoma cells expressing no or low MAGE-A levels (gray lines) and high MAGE-A levels (black lines) were treated with doses of ET as indicated. Cell viability was calculated by MTT assay 72 h after treatment. The experiment was repeated two times with similar results.