Porphyrin synthesis and ABCB6 expression in K562 cells.
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A. ABCB6 protein expression is upregulated in parental K562 cells upon chemically induced erythroid differentiation. K562 cells were cultured for 72 h with 150 nM Gleevec or 50 µM hemin, the number of viable cells was determined by trypan-blue staining. Hemoglobin levels were quantified by benzidine staining. Error bars represent SD of at least 5 experiments. ABCB6 expression is revealed by the ABCB6-567 monoclonal antibody (60 ug total protein, 72 hours post-induction). Actin is shown as loading control. B. Baseline PPIX-fluorescence of K562 cells is not influenced by ABCB6 levels. PPIX fluorescence was measured by flow cytometry as explained in the Methods section, ABCB6 expression of the cells was revealed by the anti human ABCB6-567 monoclonal antibody (actin is shown as loading control). Error bars represent SD of at least 3 experiments. Parental K562 cells (lane 1); K562 cells stably transfected with a non-target control shRNA (lane 2) or ABCB6 shRNA construct (lane 3); K562 cells stably overexpressing wild-type ABCB6 (lane 4) or a non-functional mutant variant of ABCB6 (ABCB6KM, lane 5). C. Downregulation of ABCB6 does not impede induced heme synthesis of K562 cells. K562 cells stably transfected with an shRNA construct targeting ABCB6 or a scrambled control were cultured for 72 h with 150 nM imatinib or 50 µM hemin, hemoglobin levels were quantified by benzidine staining. Error bars represent SD of at least 5 experiments.