JCS026351 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Endogenous ANKRD11 colocalizes with p53. MCF-10A cells were treated with mitomycin C (20 µg/ml) for 6 hours and immunostained with anti-ANKRD11 and anti-p53 (Ab-2) antibodies. The relative p53 protein levels in these cells with up to 6 hours of mitomycin C treatment was determined by western blot analysis using an anti-p53 antibody. β-actin was used as a loading control.

• Supplemental Figure S2 -

  Fig. S2. ANKRD11-myc cofractionates with p53 and PML. Extracts of MCF-7 cells stably expressing GFP-ANKRD11-myc were sedimented in a 10-40% glycerol gradient. Protein complexes from 24 gradient fractions were resolved on SDS-PAGE and western blotted using anti-myc, anti-p53 and anti-PML antibodies.

• Supplemental Figure S3 -

  Fig. S3. The minimal region of ANKRD11 required for p53 interaction. Findings from in vivo and in vitro interaction data (Fig. 2A-D) are presented as a schematic diagram illustrating that residues 144-313 is the minimal region of ANKRD11 that interacts with p53.

• Supplemental Figure S4 -

  Fig. S4. Restoration of ANKRD11 expression increases FAS and NOXA expression in MCF-7 and MB-468 cell lines, respectively. FAS and NOXA mRNA levels were determined in cultures stably expressing GFP-ANKRD11 or the negative control cell cultures (as described in Fig. 4A) using real-time RT-PCR analysis. Previous studies have shown that MCF-7 cells do not synthesize NOXA in response to etoposide treatment, despite induction of wild-type p53 (Yakovlev et al., 2004).

• Supplemental Figure S5 -

  Fig. S5. ANKRD11 upregulates p21^waf1 expression in a p53-dependent manner. Saos-2 cells stably-expressing GFP-ANKRD11 (Saos-2-ANKRD11) or parental Saos-2 cells (Saos-2) were established from single colonies after retroviral transduction and selected in geneticin. Total ANKRD11 expression (endogenous plus exogenous) in the Saos-2-ANKRD11 derivative was compared with the levels of endogenous ANKRD11 expressed in the parental Saos-2 cell line (upper left panel). p21^waf1 mRNA levels were determined in these cell lines following transfection with increasing amounts of a p53 expression construct (lower panels). The relative p53 expression levels in these cells were determined by western blot analysis (upper right panel).

• Supplemental Figure S6 -

  Fig. S6. Restoration of ANKRD11 in the MCF-7 and MB-468 breast cancer cell lines reduces their oncogenic properties. (A) Restoration of ANKRD11 expression reduced the clonogenic properties of the MCF-7 and MB-468 breast cancer cell lines. The number of colonies formed on plastic of the cultures stably expressing GFP-ANKRD11 or the negative control cell cultures as described in Fig. 4A was determined. Data are represented as mean ± s.e.m. of triplicate experiments. (B) Restoration of ANKRD11 expression reduced the proliferation of breast cancer cell lines. The proliferation of cultures stably expressing GFP-ANKRD11 or the negative control cell cultures as described in Fig. 4A were determined. Data are represented as mean ± s.e.m. of triplicate experiments.

• Supplemental Figure S7 -

  Fig. S7. Restoration of expression of ANKRD11 had no effect on p53 acetylation at Lys373. Total p53 and acetyl-lysine p53 (Lys373) protein levels were detected in MCF-7, MB-468 and MB-231 cultures stably expressing GFP-ANKRD11 or the negative control cell cultures previously described (Fig. 4A) through western blot analysis using the appropriate antibodies. MCF-7-ANK-1 was cultured in the presence or absence of mitomycin C for 6 hours and is presented as a representative MCF-7 derivative from Fig. 4A.