Supplemntary Figure Legends
Supplementary Figure 1 Clioquinol inhibits the proteasome through a copperdependent mechanism.
Cellular proteins were extracted from MDAY-D2, OCI-M2, OCI-AML2, K562, LP1, and
JJN3 cells with lysis buffer. Equal protein amounts were treated with increasing
concentrations of Clioquinol (CQ), CuCl2 (Cu), or an equimolar concentration of CQ and
Cu for two hours in assay buffer. After incubation, the preferential chymotrypsin-like
enzymatic activity wsa measured as described in Fig 1.
Supplementary Figure 2 Copper-binding of Clioquinol analogues.
The method of continuous variance was used to determine the conditional
stability
constants for copper(II) complexes of A) Clioquinol in DMSO, and B) chloroquine in
water (total molar concentration = 5x10-4 M).
Supplementary Figure 3 Clioquinol-induced cell death is copper dependent
A) OCI-MY5 myeloma cells were treated with CQ (20 μM) with and without Cu (20
μM), or the strong copper chelator tetrathiomoylbdate (TM) (20 μM). Twentyfour hours after incubation, apoptosis was measured by Annexin V and PI
staining and flow cytometry. B) Primary acute myeloid leukemia (AML) blasts (n
= 3) or normal peripheral blood stem cells (PBSC) (n = 3) were obtained from the
peripheral blood of consenting patients with AML or donors of PBSC for
allotransplantation, respectively.
Mononuclear cells were isolated by Ficol
separation and treated with increasing concentrations of Clioquinol (CQ) with or
without an equimolar concentration of CuCl2 (Cu). Twenty-four hours after
incubation, cell viability and apoptosis was measured by Annexin V and PI
staining and flow cytometry.