Extended Data Figure 10. NCOA4 mediates autophagic delivery of ferritin to lysosomes to control iron homeostasis.

a, Immunostaining of 8988T cells transfected with luciferase control siRNA or two independent siRNAs to NCOA4 and subjected to DFO chelation in the presence of lysosomal protease inhibitors for 9 hours. Scale bar, 20 μm. b, Immunostaining of 8988T cells transfected with luciferase control siRNA or two independent siRNAs to HERC2 and subjected to DFO chelation in the presence of lysosomal protease inhibitors for 9 hours. Scale bar, 20 μm. c, 8988T cells were transfected with luciferase control siRNA or two independent siRNAs to HERC2. Lysates were immunoblotted using antibodies to HERC2 and ACTB. d, siRNA-mediated knockdown of NCOA4 in U2OS, IMR90 and, 8988T cells leads to increase in IRP2, FTH1, and TFRC levels. Cells were transfected with luciferase control siRNA or two independent siRNAs to NCOA4. Lysates were immunoblotted using antibodies to NCOA4, IRP2, TFRC, FTH1, and ACTB. Light and dark exposures are shown for TFRC. e, NCOA4 expression levels are affected by iron levels and NCOA4 levels affect ferritin levels in response to iron load. 8988T cells stably expressing either a control MSCV empty vector or mouse NCOA4 were cultured in the presence of FAC for the indicated times. Cells were lysed and analyzed by immunoblotting with antibodies to NCOA4, FTH1 and ACTB as a loading control. A non-specific band migrates just below the NCOA4 specific band.