Figure 3.

Loss of LcytB and Steap3 is additive in limiting the export of lysosomal iron. (A) Steap3 was mutated in LcytB #2 as described in Figure 2. qRT-PCR analysis was performed on WT and DKD #1 and DKD #2 clones using Steap3 and Actin primers. Data were normalized to a WT level of 1.0. (n > 3-7 biological replicates). (B) Crude membrane preparations were performed for WT, DKD #1, DKD #2, and Steap3 #2, and western blot analysis for Steap3 and VDAC was performed. VDAC was used as a membrane loading control. A representative blot with 2 biological replicates is shown. (C) Western blots were quantified using Fiji Image J using VDAC as a membrane loading control. Data were normalized to a WT level of 1.0 (n = 4 biological replicates). (D) Cytosolic ferritin levels were determined in cells incubated in the presence or absence of CF as in Figure 2B. Data were normalized to a WT cytosolic ferritin level (–CF) of 1.0 (n ≥ 6 biological replicates). (E) WT cells were treated with or without CF as in Figure 1B and fixed. Steap3 and Lamp2 localization were detected by confocal immunofluorescence microscopy. A representative field is shown for –CF +CF. Arrows denote areas of Steap3 and Lamp2 colocalization in CF-loaded cells. (F) Data from 5 biological replicate slides were quantified, and the data were displayed as percentage of cells showing Steap3 and Lamp2 colocalization. More information on procedures is provided in “Materials and methods.” Error bars represent SEM. *P < .05; **P < .01; ***P .001; ****P < .0001.