Supplementary Materials

Supplementary Materials for:

A Network of Substrates of the E3 Ubiquitin Ligases MDM2 and HUWE1 Control Apoptosis Independently of p53

Manabu Kurokawa,* Jiyeon Kim, Joseph Geradts, Kenkyo Matsuura, Liu Liu, Xu Ran, Wenle Xia, Thomas J. Ribar, Ricardo Henao, Mark W. Dewhirst, Wun-Jae Kim, Joseph E. Lucas, Shaomeng Wang, Neil L. Spector, Sally Kornbluth*

*Corresponding author. E-mail: manabu.kurokawa@dartmouth.edu (M.K.); sally.kornbluth@duke.edu (S.K.)

This PDF file includes:

  • Fig. S1. p53 status of the cell lines used in this study.
  • Fig. S2. MI-219 can reverse lapatinib resistance.
  • Fig. S3. SRC abundance in the cell lines used in this study.
  • Fig. S4. The SRC inhibitor dasatinib does not affect MDM2 protein abundance.
  • Fig. S5. BT474 cells with acquired lapatinib resistance prevent mitochondrial cytochrome c release in response to lapatinib treatment.
  • Fig. S6. Mcl-1 abundance increases in SKBR3 cells with acquired lapatinib resistance following lapatinib treatment.
  • Fig. S7. Mcl-1 is stabilized in lapatinib-resistant SKBR3 cells.
  • Fig. S8. Ubiquitin aldehyde does not prevent the failure to degrade Mcl-1 in the resistant cells.
  • Fig. S9. Unaltered abundance of Apaf-1.
  • Fig. S10. CK2-mediated phosphorylation of HSP90β at Ser226 and Ser255.
  • Fig. S11. PP5 is stabilized in lapatinib-resistant AU565 cells.
  • Fig. S12. Biochemical purification of the PP5-targeted E3 ligase.
  • Fig. S13. Overexpression of HUWE1 leads to reduced abundance of PP5 and Mcl-1.
  • Fig. S14. CAS degradation in lapatinib-resistant AU565 cells.
  • Fig. S15. Enhanced CAS polyubiquitylation in lapatinib-resistant SKBR3 cells.
  • Fig. S16. Schematic diagram of the CAS protein sequence.
  • Fig. S17. Densitometric analysis of HUWE1 and MDM2 abundance after lapatinib treatment.
  • Fig. S18. Schematic representation of HUWE1 protein and the amino acid sequence surrounding the MDM2 binding motif.
  • Fig. S19. HUWE1 and MDM2 interact in cells.
  • Fig. S20. Densitometric analysis of the abundance of HUWE1, MDM2, CAS, PP5, and Mcl-1 upon lapatinib–Nutlin-3a cotreatment.
  • Fig. S21. Nutlin-3 can reverse lapatinib resistance in rAU565 xenografts.
  • Table S1. Biomarker status of breast cancer cell lines used in this study.
  • References (44, 45)

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Citation: M. Kurokawa, J. Kim, J. Geradts, K. Matsuura, L. Liu, X. Ran, W. Xia, T. J. Ribar, R. Henao, M. W. Dewhirst, W.-J. Kim, J. E. Lucas, S. Wang, N. L. Spector, S. Kornbluth, A Network of Substrates of the E3 Ubiquitin Ligases MDM2 and HUWE1 Control Apoptosis Independently of p53. Sci. Signal. 6, ra32 (2013).

© 2013 American Association for the Advancement of Science