Figure 3.
The absence of CYB561A3 will cause a P3HR-1 iron starvation response. (A) Volcano plot of differentially expressed genes in P3HR-1 twelve days after control or CYB561A3 sgRNA expression (n = 3 replicates). Selected genes upregulated (red) or downregulated (blue) by CYB561A3 depletion are highlighted. (B) Gene Ontology enrichment analysis of significantly upregulated cellular components in RNA-sequencing analysis of P3HR-1 cells with CYB561A3 vs control sgRNA expression. (C) Gene Ontology enrichment analysis of significantly downregulated biological processes in RNA-sequencing analysis of P3HR-1 cells with CYB561A3 vs control sgRNA expression. (D) Volcano plot analysis of log2 fold-change messenger RNA abundances in Mutu III vs Mutu I (y-axis) and in P3HR-1 with CYB561A3 vs control sgRNA expression (x-axis). (E) Immunoblot analysis of whole-cell lysates (WCL) from P3HR-1 (top) and GM12878 (bottom) at 10 days’ post-control or CYB561A3 sgRNA expression for the indicated proteins. (F) Mean ± SD values of quantitative polymerase chain reaction analysis of the indicated messenger RNA expression in P3HR-1 cells 10 days after control or CYB561A3 sgRNA expression from n = 3 replicates. (G) Immunofluorescence analysis of TFRC expression in P3HR-1 ten days after control or CYB561A3 sgRNA expression. Scale bar, 10 μm. Representative images from n = 2 replicates are shown. (H) Immunoblot analysis of WCL from P3HR-1 with control green fluorescent protein (GFP) or CYB561A3R (CYBR) cDNA 7 days’ post-sgRNA expression. (I) Mean ± SD values from P3HR-1 cells with control (black) or CYB561A3 (gray) sgRNA and cultured in the indicated concentration of ferrous citrate. Ferric citrate or phosphate-buffered saline was added 4 days’ post-sgRNA expression, and fold change was measured over the next 3 days. (J) Immunoblot analysis of WCL from P3HR-1 that expressed control or CYB561A3 sgRNA for 7 days and cultured in phosphate-buffered saline or ferrous citrate for days 4 to 7. Panels E, H, and J show representative blots from n = 3 replicates. *P < .05; **P < .01; ***P < .001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ns, nonsignificant (unpaired Student t test).

The absence of CYB561A3 will cause a P3HR-1 iron starvation response. (A) Volcano plot of differentially expressed genes in P3HR-1 twelve days after control or CYB561A3 sgRNA expression (n = 3 replicates). Selected genes upregulated (red) or downregulated (blue) by CYB561A3 depletion are highlighted. (B) Gene Ontology enrichment analysis of significantly upregulated cellular components in RNA-sequencing analysis of P3HR-1 cells with CYB561A3 vs control sgRNA expression. (C) Gene Ontology enrichment analysis of significantly downregulated biological processes in RNA-sequencing analysis of P3HR-1 cells with CYB561A3 vs control sgRNA expression. (D) Volcano plot analysis of log2 fold-change messenger RNA abundances in Mutu III vs Mutu I (y-axis) and in P3HR-1 with CYB561A3 vs control sgRNA expression (x-axis). (E) Immunoblot analysis of whole-cell lysates (WCL) from P3HR-1 (top) and GM12878 (bottom) at 10 days’ post-control or CYB561A3 sgRNA expression for the indicated proteins. (F) Mean ± SD values of quantitative polymerase chain reaction analysis of the indicated messenger RNA expression in P3HR-1 cells 10 days after control or CYB561A3 sgRNA expression from n = 3 replicates. (G) Immunofluorescence analysis of TFRC expression in P3HR-1 ten days after control or CYB561A3 sgRNA expression. Scale bar, 10 μm. Representative images from n = 2 replicates are shown. (H) Immunoblot analysis of WCL from P3HR-1 with control green fluorescent protein (GFP) or CYB561A3R (CYBR) cDNA 7 days’ post-sgRNA expression. (I) Mean ± SD values from P3HR-1 cells with control (black) or CYB561A3 (gray) sgRNA and cultured in the indicated concentration of ferrous citrate. Ferric citrate or phosphate-buffered saline was added 4 days’ post-sgRNA expression, and fold change was measured over the next 3 days. (J) Immunoblot analysis of WCL from P3HR-1 that expressed control or CYB561A3 sgRNA for 7 days and cultured in phosphate-buffered saline or ferrous citrate for days 4 to 7. Panels E, H, and J show representative blots from n = 3 replicates. *P < .05; **P < .01; ***P < .001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ns, nonsignificant (unpaired Student t test).

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