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Blood, Vol. 111, Issue 9, 4690-4699, May 1, 2008
Targeting the p27 E3 ligase SCFSkp2 results in p27- and Skp2-mediated cell-cycle arrest and activation of autophagy
Blood Chen et al.
111: 4690
Supplemental materials for: Chen et al
Files in this Data Supplement:
- Figure S1 (JPG, 62.4 KB)
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(A) Image J software was used to quantify the protein bands in Fig.1C. Both p27 and phosphop27 levels were normalized for loading using  -Actin, and the ratio at time 0 was arbitrarily set at 1.0. These data indicated that both Cpd A and MG132 treatment prolonged the half-life of p27 and phospho-p27. (B) MM1.S cells treated with CpdA were examined for their content of several SCF substrate proteins and cell cycle-related proteins by Western blotting. Substrates of SCFSkp2 such as p21, p27, and p57 were stabilized, but the abundance of components of this SCF were not changed, nor were substrates of other E3 ligases such as c-Jun. (C) MM1.S cells were treated with DMSO, CpdA, or bortezomib for 24 hours, and whole cell lysates were subjected to Western blotting using anti-phospho-HSP-27, HSP-70, and HSP-90 antibodies. Unlike bortezomib, which activated the heat shock protein response, CpdA did not increase the abundance of HSP-70 or -90 nor the phosphorylation of HSP-27. (D) MM1.S cells were pre-incubated with vehicle or 50 µM PAN-caspase inhibitor for two hours, and then treated for 24-hours with vehicle, or the indicated concentrations of CpdA or bortezomib. Cell viability was determined using the WST-1 assay, and is shown as a mean value ± SD from triplicate experiments. Caspase inhibition protected MM1.S cells from bortezomibmediated cell death, but not from CpdA-induced autophagy. (E) MM1.S treated under conditions indicated above were analyzed for their content of LC3 forms-I and –II by Western blotting, with -Actin serving as a loading control. Consistent with the activation of autophagy, CpdA induced appearance of MAP-LC3-form II.
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