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Blood, Vol. 113, Issue 19, 4667-4676, May 7, 2009
Targeted inhibition of the immunoproteasome is a potent strategy against models of multiple myeloma that overcomes resistance to conventional drugs and nonspecific proteasome inhibitors
Blood Kuhn et al.
113: 4667
Supplemental materials for: Kuhn et al
Files in this Data Supplement:
- Table S1. Combination indices of IPSI-001 and dexamethasone in ANBL-6 cells (PDF, 316 KB)
- Figure S1. IPSI-001 decreases cell viability (JPG, 39.7 KB)
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(A) ANBL-6 cells were measured for apoptotic cell content after treatment with IPSI-001 for 24 hours. (B) RPMI 8226 cells were treated with increasing concentrations of IPSI-001, bortezomib, epoxomicin, and lactacystin for 24 hours than assayed for anti-proliferative effects using WST-1 reagent.
- Figure S2. IPSI-001 preferentially induces loss of mitochondrial membrane potential in lymphoid-derived KAS-6/1 cells over HT-29 solid tumor cells (JPG, 68.1 KB)
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KAS-6/1 myeloma and HT-29 colon cancer cells were incubated with indicated concentrations of IPSI-001 or bortezomib for 24 hours. The cationic dye, JC-1, was then added to determine loss of membrane potential (ΔΨ). In live cells the dye accumulates in the mitochondria as aggregates (Red), whereas in dead cells it would remain in the cytosol as monomers (Green). Therefore, loss of red fluorescence correlates with mitochondrial membrane collapse. Magnification 20×.
- Figure S3. Proteasome subunit expression in cell lines (JPG, 37.9 KB)
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Western blot analysis of constitutive proteasome (19S S6′; β5) and immunoproteasome (11S Reg α; β1i) subunits in solid and hematologic tumor cell lines. Solid tumors in lanes 1–6 (SK-N-MC, LNCaP, PC-3, Hs294T, BT474 and HeLa) and hematologic cells in lanes 7–11 (AML193, ANBL-6, KAS-6/1, RPMI 8226, and SupB8) were lysed and expression of proteasome subunits and regulatory particles were assessed by Western blot. HSC-70 is used as a loading control.
- Figure S4. 8226.BR cells are resistant to bortezomib (JPG, 28.1 KB)
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8226.BR cells are resistant to concentrations of bortezomib up to 50 nM for 24 hours.
- Figure S5. IPSI-001 synergizes with dexamethasone (JPG, 20.4 KB)
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ANBL-6 cells were treated with ISPI-001 alone, dexamethasone alone, or simultaneously with both drugs for 48 hours. Compared with dexamethasone or IPSI-001 alone, the combination demonstrated a greater anti-proliferative effect. Data shown are combined from two independent experiments.
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