CYB561A3 KO depletes Burkitt cell ferrous iron. (A) Confocal microscopy analysis of FerroOrange-stained P3HR-1 or GM12878 expressing control or CYB561A3 sgRNAs for the indicated days. Shown at right are mean ± standard deviation (SD) values from n = 3 replicates of fluorescence-activated cell sorter mean FerroOrange signals. Scale bar, 10 μm. (B) Confocal analysis of RPA-stained P3HR-1 expressing control or CYB561A3 sgRNAs for 10 days. Shown at right are mean ± SD values from n = 3 replicates of fluorescence-activated cell sorter mean RPA values. (C) Aconitase activity assay from P3HR-1 cells with control or CYB561A3 sgRNAs and grown with the indicated amount of ferric citrate supplementation. Bar plots show mean ± SD aconitase activity. (D) Immunoblot analysis of OXPHOS subunits or tubulin load control from whole-cell lysates (WCL) of P3HR-1 with control or CYB561A3 sgRNAs and grown with the indicated amount of ferric citrate supplementation. (E) Immunoblot analysis of lipoic acid, which is conjugated to PDH-E2 and KGDH-E2 subunits or tubulin load control from WCL of P3HR-1 with control or CYB561A3 sgRNAs and grown with the indicated amount of ferric citrate supplementation. (F) Fifty percent inhibitory concentration (IC₅₀) analysis of P3HR-1 after control (black) or CYB561A3 sgRNAs #1 (red) or #2 (green) expression and treated with the indicated erastin concentrations from days 9 to 12. Shown are mean ± SD values and calculated IC₅₀ values from n = 3 replicates. (G) Immunoblot analysis of WCL from P3HR-1 and GM12878 at 12 days’ post-control or CYB561A3 sgRNA expression. Panels D, E, and G show representative blots from n = 3 replicates. *P < .05 by 1-way analysis of variance with Bonferroni posttest. **P < .01, ***P < .001 (unpaired Student t test). GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HIF1α, hypoxia inducible factor 1 subunit α; ns, nonsignificant.