Fig. 3. H-Ras G12V-transformed MCF10A are addicted to high 26S proteasome levels.

a Schematic description of the experimental strategy. MCF10A stably expressing doxycycline-inducible PSMD1 shRNA were further subjected to transformation with H-Ras G12V. PSMD1 KD was induced by the addition of 1 μg/ml doxycycline for 72 h. b Protein content in the MCF10A naive and H-Ras G12V-transformed cells, in the presence or absence of PSMD1 shRNA induction, was analyzed by immunoblot. Quantification of the amount PSMD1, normalized to the amount of actin, is presented. c The 26S and 20S proteasomal complex levels were analyzed by native gel electrophoresis as described in Fig. 1c. d The levels of the 26S and 20S proteasome activity were determined as in Fig. 1c. Equal amounts of total protein, determined by Bradford assay, were loaded onto native gels (actin loading control is represented). e Cell proliferation rate was analyzed using the XTT assay. Naive or Ras-transformed MCF10A cells harboring a doxycycline-inducible PSMD1 shRNA were either doxycycline treated to induce PSMD1 shRNA expression or left untreated, and cell proliferation was followed daily. XTT at the seeding day was taken as 100% in measuring relative cell growth