Figure 4.
CYB561A3 ferrireductase activity is critical for Burkitt B-cell iron acquisition. (A) Mean ± standard deviation (SD) fold-change live cell values from n = 3 replicates of P3HR-1 with control (black) or CYB561A3 sgRNA (gray) expression and cultured in the indicated concentration of ascorbic acid. Phosphate-buffered saline or ascorbic acid was added at days 4 to 9 post-sgRNA expression. Fold change was measured from days 4 to 9. (B) Immunoblot analysis of whole-cell lysates from P3HR-1 expressing control or CYB561A3 sgRNAs and cultured in the indicated ascorbic acid concentrations, as in panel A. (C) Fluorescence-activated cell sorter analysis of plasma membrane TFRC expression in P3HR-1 expressing control (pink) or CYB561A3 (blue) sgRNAs. Phosphate-buffered saline or ascorbic acid was added from days 4 to 9 post-sgRNA expression, at which time fluorescence-activated cell sorter was done. Representative histograms from n = 3 replicates are shown. (D) Schematic model of CYB561A3. Red stars represent selected amino acids indicated in CYB561A3 electron transport. (E) Confocal microscopy analysis of P3HR-1 lysosomal marker LAMP1, antihemagglutinin (HA) tagged CYB561A3 WT, or indicated alanine point mutant stained for HA, and Hoechst-stained nuclei merge image. Scale bars, 10 μm. Representative images from n = 3 replicates are shown. (F) Mean ± SD values from n = 3 replicates of P3HR-1 with control (black) or CYB561A3 (gray) sgRNA and the, cDNA encoding control green fluorescent protein (GFP) or CYB561A3 WT or alanine point mutants. Fold change was measured from days 4 to 8 post-sgRNA expression. (G) Immunoblot analysis of whole-cell lysates from P3HR-1 expressing the indicated sgRNA and cDNA, as in panel F, at day 8 post-sgRNA expression. (H) CYB561 overexpression does not compensate for loss of CYB561A3. Stable expression of HA-epitope–tagged CYB561 vs CYB561A3R cDNAs was validated by confocal analysis of P3HR-1 stained with anti-HA antibody (left). Control or CYB561A3 sgRNAs were then expressed, as indicated. Mean ± SD fold-change live cell values from n = 3 replicates of cells with the indicated sgRNA and cDNA expression. Panels B and G show representative blots from n = 3 replicates. *P < .05; **P < .01; ***P < .001. ns, nonsignificant (unpaired Student t test).

CYB561A3 ferrireductase activity is critical for Burkitt B-cell iron acquisition. (A) Mean ± standard deviation (SD) fold-change live cell values from n = 3 replicates of P3HR-1 with control (black) or CYB561A3 sgRNA (gray) expression and cultured in the indicated concentration of ascorbic acid. Phosphate-buffered saline or ascorbic acid was added at days 4 to 9 post-sgRNA expression. Fold change was measured from days 4 to 9. (B) Immunoblot analysis of whole-cell lysates from P3HR-1 expressing control or CYB561A3 sgRNAs and cultured in the indicated ascorbic acid concentrations, as in panel A. (C) Fluorescence-activated cell sorter analysis of plasma membrane TFRC expression in P3HR-1 expressing control (pink) or CYB561A3 (blue) sgRNAs. Phosphate-buffered saline or ascorbic acid was added from days 4 to 9 post-sgRNA expression, at which time fluorescence-activated cell sorter was done. Representative histograms from n = 3 replicates are shown. (D) Schematic model of CYB561A3. Red stars represent selected amino acids indicated in CYB561A3 electron transport. (E) Confocal microscopy analysis of P3HR-1 lysosomal marker LAMP1, antihemagglutinin (HA) tagged CYB561A3 WT, or indicated alanine point mutant stained for HA, and Hoechst-stained nuclei merge image. Scale bars, 10 μm. Representative images from n = 3 replicates are shown. (F) Mean ± SD values from n = 3 replicates of P3HR-1 with control (black) or CYB561A3 (gray) sgRNA and the, cDNA encoding control green fluorescent protein (GFP) or CYB561A3 WT or alanine point mutants. Fold change was measured from days 4 to 8 post-sgRNA expression. (G) Immunoblot analysis of whole-cell lysates from P3HR-1 expressing the indicated sgRNA and cDNA, as in panel F, at day 8 post-sgRNA expression. (H) CYB561 overexpression does not compensate for loss of CYB561A3. Stable expression of HA-epitope–tagged CYB561 vs CYB561A3R cDNAs was validated by confocal analysis of P3HR-1 stained with anti-HA antibody (left). Control or CYB561A3 sgRNAs were then expressed, as indicated. Mean ± SD fold-change live cell values from n = 3 replicates of cells with the indicated sgRNA and cDNA expression. Panels B and G show representative blots from n = 3 replicates. *P < .05; **P < .01; ***P < .001. ns, nonsignificant (unpaired Student t test).

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