CYB561A3 KO causes Burkitt cell cycle arrest and lysosomal and mitochondrial damage. (A) Cell cycle analysis of P3HR-1 and GM12878 nine days after control or CYB561A3 sgRNA expression. Lower panel, mean ± standard deviation (SD) values from n = 3 replicates are shown. (B) Fluorescence-activated cell sorter forward scatter (FSC; cell size) and side scatter (SSC; internal complexity) mean ± SD values from n = 3 replicates obtained 12 days after P3HR-1 control or CYB561A3 sgRNA expression. (C) Fluorescence-activated cell sorter analysis of mitochondrial reactive oxygen species signals detected by mitoSOX in P3HR-1 cells 12 days after control or CYB561A3 sgRNA expression, with mean ± SD values from n = 3 replicates. (D) Immunofluorescence analysis of P3HR-1 transfected with a green fluorescent protein (GFP)-tagged CYB561A3 expression vector and stained with LysoTracker to visualize lysosomes. Shown below is the merge, together with Hoechst-stained nucleus. Image is representative of n = 3 replicates. (E) Acridine Orange (1 μg/mL) staining of lysosomes in P3HR-1 cells 12 days after control or CYB561A3 sgRNA expression. Scale bar indicates 10 μm. Representative images from n = 3 replicates are shown. (F) EM images of P3HR-1 twelve days after control or CYB561A3 sgRNA expression. Arrows indicate representative mitochondria (red), lysosomes (white), and autophagosomes (blue). White scale bar sizes are noted at bottom right. (G) Seahorse analysis of P3HR-1 and GM12878 cell oxygen consumption rate (OCR) 12 days after control (black) or CYB561A3 (red) sgRNA expression. Time points of oligomycin (3.5 µM), CCCP (2 µM), and piericidin A (100 nM) addition are indicated. Mean ± SD from n = 3 replicates are shown. **P < .01; ***P < .001. ns, nonsignificant (unpaired Student t test).