Modulation of Hh signaling modifies doxorubicin incorporation in leukemia cells. Modulation of Hh signaling modifies doxorubicin incorporation in leukemia cells. A, Ptc is expressed in K562 cells. Total protein extracts (200 μg) from K562 cells pretreated or not with 30 nmol/L ShhN (3 hours), 10 μmol/L CPN (48 hours) or 10 μg/mL lovastatin (24 hours) were immunoblotted. The signals of Ptc and loading control were quantified using Image J software. The histogram is the mean ± SEM of 3 independent experiments (***P < 0.0005). B, modulators of Hh signaling act on doxorubicin accumulation in K562 cells. K562 cells pretreated or not with the Hh signaling agonist SAG (30 nmol/L), the Hh signaling antagonist CPN (10 μmol/L), the ABC transporters inhibitor Verapamil (5 μmol/L) were incubated for 30 minutes in physiologic buffer containing 10 μmol/L doxorubicin supplemented or not with 30 nmol/L ShhN before rinsing and fluorescence measurement. Values reported are the mean ± SEM of 3 independent experiments. C, CPN pretreatment or ShhN presence increases doxorubicin incorporation in K562 cells. K562 cells pretreated or not with CPN (10 μmol/L) were added to spectrofluorimeter cuvettes containing 1 μmol/L doxorubicin in physiologic buffer supplemented or not with ShhN protein (30 nmol/L). Time-dependent doxorubicin fluorescence quenching was recorded simultaneously in the 3 cuvettes. The quenching slopes were calculated for 3 independent experiments and mean ± SEM were reported (*P < 0.05). D, lovastatin treatment increases doxorubicin incorporation in K562 cells. K562 cells pretreated or not with lovastatin (10 μg/mL) were added to spectrofluorimeter cuvettes containing 1 μmol/L doxorubicin in physiologic buffer. Time-dependent doxorubicin fluorescence quenching was recorded simultaneously in both cuvettes. The quenching slopes were calculated for 3 independent experiments and mean ± SEM were reported (*P < 0.05). Michel Bidet et al. Mol Cancer Res 2012;10:1496-1508 ©2012 by American Association for Cancer Research