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1 DOI: /ncb1875 Figure S1 (a) The 79 surgical specimens from NSCLC patients were analysed by immunohistochemistry with an anti-p53 antibody and control serum (data not shown). The normal bronchi served as the negative control. Scale bars represent 100 µm. AD, adenocarcinoma; SCC, squamous cell carcinoma. (b, c) p53 overexpression decreases the level of Slug protein but not mrna. H1299 cells were transfected with increasing concentrations of p53 and a constant amount of HA-Slug. At 36 h post-transfection, cell lysates were analysed by immunoblotting (IB) (b) or RT-PCR analysis (c). β-actin protein and Gβ-like mrna, respectively, served as controls for equal loading. Numbers below the row of the RT-PCR indicate the densitometric values normalised with the relative Gβ-like value. (d) RNAi knockdown of p53 reverses p53-mediated Slug downregulation. H1299 cells were cotransfected with HA-Slug, p53 and psuper-p53-sirna or plk0.1-p53- shrna in different combinations and protein expression was evaluated by immunoblotting 36 h post-transfection. Apoptosis was quantified by FACS analysis and the percentage of sub-g1 cells is shown. (e) MDM2 promotes Slug ubiquitination in vivo. p53 -/- Mdm2 -/- MEFs were infected with Lenti- MDM2 for 48 h, and then treated with MG132. After immunoprecipitation of Slug with α-slug, the ubiquitinated-slug complexes were detected by immunoblotting using an α-ub antibody. (f) Knockdown of MDM2 stabilises Slug protein. HFBs transfected with control or Mdm2-siRNA oligonucleotides were lysed and analysed by immunoblotting 72 h after transfecting. 1

2 Figure S2 (a, b) Interactions of endogenous p53, MDM2, and Slug in HEK293 and HFB cells. Cells were treated with 10 µm MG132. After 6 h, cell lysates were immunoprecipitated with α-p53 (DO-1), two independent α-mdm2 (SMP-14 and/or Ab-3), or mouse IgG before immunoblotted with specific antibodies (SMP-14, α-p53 or α-slug). Right panel: the protein bands detected in co-ip assays were characterised by specific sirnas. (c) Loss of p53 expression reduces the levels of Slug ubiquitination. p53 +/+ or p53 -/- MEFs were treated with 10 µm MG132 for 6 h. After immunoprecipitation of Slug with α-slug, the ubiquitinated-slug complexes were detected by immunoblotting using an α-ub antibody (Pierce). (f) p53 enhances the MDM2-mediated degradation of Slug. p53 -/- Mdm2 -/- MEFs stably transduced with Lenti-MDM2 or control Lenti-eGFP were infected with differential doses of Ad-p53. After 48 h, cells were lysed and analysed by immunoblotting with indicated antibodies. Numbers below the respective panels of the immunoblots indicate the densitometric values normalised with the relative control (as indicated) value. 2

3 Figure S3 (a) Knockdown of Slug in CL1-5 cells by Slug-siRNA oligonucleotides increases E-cadherin expression and decreases cell invasiveness. (b) The effects of mutant p53 silencing on MDM2, Slug, E-cadherin, and cancer cell invasiveness can be observed by an independent p53-shrna (sh-p53-2). (c, d) The effects of mutant p53 silencing on MDM2, Slug, E-cadherin, and cancer cell invasiveness can be observed in other mutant p53-expressing cancer cell lines, MDA-MB-231 (c) and SW620 (d). Data are shown as mean ± SEM from three independent experiments (n=3). (e, f) p73 interacts with p53r280k in MDA-MB-231 cells (e) or with p53r273h in SW620 cells (f). (g, h) Correlation of p53 activation, Slug expression, and p21 and Mdm2 induction with apoptosis in UV-irradiated HFB (p53 +/+ ) cells. HFB cells were exposed to 50 J m -2 of UV radiation. At different time points after UV radiation, cells were lysed and analysed for expression of p53, MDM2, p21 and Slug by immunoblotting (g) and RT-PCR analysis (h). Phosphorylation of p53 at Ser46 was also evaluated by immunoblotting. β-actin served as an internal control for both immunoblotting and PCR analyses. Numbers below the RT-PCR indicate the densitometric values normalised with the relative β-actin value. Apoptotic cells were detected using an Annexin V-PI FACS-based assay. The percentage of PI-negative, annexin V-positive cells at different time points after UV radiation is indicated. 3

4 Figure S4 Full scans of key Western data shown in the regular Figures 1c, 1e, 1f, 3e, 3f, 3g, 4b, 4c, 4e, 6a, 6b, 6d, 6e, and Supplementary Information, Figure S2d. 4

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9 Supplementary discussion The phosphorylation and ubiquitin-mediated proteasomal degradation of Snail (SNAI1) has recently been described 1. Unlike SNAI1, little is known about the post-translational regulation of Slug. Vernon et al. have shown that, in Xenopus, the partner of paired (Ppa) protein, the F-box component of a modular E3 ligase, regulates the degradation of Slug in neural crest cells 2. Here we found that human Slug is a labile protein whose degradation is mediated by p53 and MDM2 in normal cells and cancer cells. Because the Slug protein plays essential functions in embryonic development and cancer progression, multiple sophisticated mechanisms are likely involved in controlling its activity. Our findings imply that the stability of Slug is normally under fine control, and dysregulation of Slug catabolism may invigorate tumour malignancy 3. It has been shown that p53, through its p53 DNA-binding domain, interacts directly with bcl-xl and bcl-2 to mediate apoptosis (ref. 32 in main text), indicating that this domain possesses functions apart from transcriptional regulation. In our study, we showed that p53 can bind to Slug via its DNA-binding domain, forming a complex with Slug and MDM2 that facilitates Slug degradation. These results corroborate a previous report that p53 guides the recognition and recruitment of HIF-1α to MDM2-ubiquitin ligase, facilitating ubiquitin-dependent degradation by the proteasome 4. In addition, we showed that an N-terminal region (amino acids 21-66) of Slug is responsible for binding p53 and MDM2 and is likely critical for Slug degradation. A more refined characterization of the interacting domains within the p53-mdm2-slug complex and a functional analysis of their contributions to Slug degradation may assist in the rational design of new cancer therapies.

10 Supplementary references 1. Peinado, H., Olmeda, D., & Cano, A. Snail, Zeb and bhlh factors in tumour progression: an alliance against the epithelial phenotype? Nat. Rev. Cancer 7, (2007). 2. Vernon, A. E. & LaBonne, C. Slug stability is dynamically regulated during neural crest development by the F-box protein Ppa. Development 133, (2006). 3. Perez-Mancera, P. A. et al. SLUG in cancer development. Oncogene 24, (2005). 4. Ravi, R. et al. Regulation of tumor angiogenesis by p53-induced degradation of hypoxia-inducible factor 1alpha. Genes Dev. 14, (2000).