Fig. 4

Chloroquine and bortezomib downregulate expression and activity of Pgp. MDA-MB-231 and JC cells were cultured for 24 h in the absence (ctrl) or presence of the lysosome inhibitor chloroquine (CQ; 1 μM) or the proteasome inhibitor bortezomib (B; 1 μM), alone or in combination. a. The relative expression of Pgp gene was measured by qRT-PCR. Data are presented as means±SD (n = 4). *p < 0.02: treated cells vs ctrl cells; °p < 0.001: CQ + B-treated cells vs CQ/B-treated cells. b. Plasma-membrane extracts were probed for Pgp or immunoprecipitated with an anti-nitrotyrosine antibody, then probed for Pgp (nitroPgp). The expression of pancadherin was used as control of equal membrane protein loading. The figure is representative of 1 out of 3 experiments. c. Pgp activity was analyzed in duplicates by a spectrophotometric assay. Data are presented as means±SD (n = 4). *p < 0.02: treated cells vs ctrl cells; °p < 0.002: CQ + B-treated cells vs CQ/B-treated cells. d. Doxorubicin efflux (i.e. the change of intracellular doxorubicin concentration per unit of time; dc/dt) was measured in triplicates by a fluorimetric assay, in cells incubated 10 min with increasing concentrations of doxorubicin to achieve the maximal velocity efflux (Vmax). Data are presented as means±SD (n = 3). *p < 0.001: treated cells vs ctrl cells; °p < 0.001: CQ + B-treated cells vs CQ/B-treated cells