Effects of RAMB treatment on the levels of polyubiquitinated proteins in HeLa cells.

<p><i>Left panel:</i> immunoblot analysis of ubiquitinated proteins in HeLa cells after 6 hours exposure with or without 10 µM RAMBs. Bortezomib was used as positive control. Equal protein loading in each lane was verified by using an antibody against GAPDH. <i>Right panel:</i> Quantification of the Ubiquitin/GAPDH ratios.</p

RAMB1 treatment prevents anchorage-dependent colony formation and synergizes with Chloroquine to kill cervical cancer cells.

<p>A. Equal numbers of SiHa and Caski cells (10³) were seeded into 6-wells plastic dishes and treated with or without RAMB1 at the indicated concentrations over a period of 10 days. Colonies were visualized by crystal violet staining. B. <i>Left panel</i>: Quantification of colony number in mock versus RAMB1 treated HeLa cells. <i>Middle panel</i>: Quantification of colony size in mock versus RAMB1 treated cervical cancer cells. <i>Right panel</i>: Representative experiment of HeLa cells (10²) which were seeded into 6-wells plastic dishes and treated with or without RAMB1 at the indicated concentration over a period of 10 days. Colonies were visualized by crystal violet staining and manually counted using an inverted microscope. <i>Inserts:</i> Representative example of colony size in mock versus RAMB1-treated cells. <i>Middle panel:</i> Quantification of colony number. <i>Right panel</i>: Quantification of colony size, representative of three independent experiments. **, P<0.02. C. CaSki cells were treated with checkerboard dilution series of RAMB1 and Chloroquine over a period of 24 hours. Cell viability was measured by XTT assay and calculated as percent of control untreated cultures. Synergy is expressed as combination index (CI).</p

Effect of RAMB treatment upon cervical cancer cell lines and primary human keratinocytes.

<p>Cultures of HPV-transformed cervical cancer cells (SiHa, CaSki and ME180) or primary human keratinocytes were treated with the indicated concentrations of RAMB1 (<i>left panel</i>) or RAMB4 (<i>right panel</i>) over a period of 48 hours. Cell viability was determined by XTT assay and plotted as a fraction of the untreated control cultures.</p

RAMB treatment fails to inhibit proteasomal activity in living cells.

<p><i>Left panel:</i> Luciferase activity in lysates of Ub-FL or FL-vector transfected cells either with or without treatment with RAMB compounds or Bortezomib were quantified in Relative Luminescence Units (RLU) and expressed as % of control. Error bars are Standard Deviation (SD) for three independent experiments. <i>Middle panel:</i> Quantification of the Ub-FL/FL ratio. <i>Right panel:</i> Luminescence activity in cell lysates derived from CaSki cervical cancer cells with or without RAMB or Botezomib treatment quantified in Relative Luminescence Units (RLUs). *, P<0.05, **, P<0.02.</p

RAMB treatment fails to inhibit the 20S proteasome proteolytic activities.

<p>Purified 20S proteasomes were treated for 30 min with or without RAMB compounds or Bortezomib, here used as positive control, at the indicated concentrations and the specific fluorogenic substrates for chymotrypsin-like, trypsin-like and peptidylglutamyl peptide hydrolyzing-like hydrolytic proteasome capacities were subsequently added. A representative example of two independent experiments is shown.</p