Figure 3.

Chloroquine impairs Hb degradation but not primary cellular Hb uptake. (a) Fluorescent Hb:Hp (Alexa 633) uptake capacity of chloroquine pretreated (30 min) and control CD163-positive HEK cells (values given as mean ± SEM of channel fluorescence from three independent experiments). No statistical difference was observed (P > 0.05). (b) CD163-positive HEK cells, with and without chloroquine pretreatment (as shown in Figure 2), were incubated for 12 hours with Hb:Hp 2 mg/mL. After removing Hb from culture medium by extensive washing, the cell samples were either immediately lysed or further incubated for 180 min before intracellular Hb-specific peptides were relatively quantified by SRM. The following three Hb peptides were monitored and are individually represented in the graph: SAVTALGK, MFLSFPTTK, and LLVVYPWTQR (see Figure 1). The Hb decay is significantly less in the chloroquine-treated cells (control 0 versus 180 min P < 0.05, chloroquine-treated 0 versus 180 min P > 0.05). Data points represent mean peptide abundance values that were determined in three independent experiments.