Fig. S3

Fig. S3 Requirement of BDH2 for Intracellular Iron Homeostasis in Mouse mIMCD Kidney Cells, Related to [Figure 4], [Figure 5] and [Figure 6]

(A) RT-PCR analysis monitoring expression of Bdh2 or, as a loading control, Gapdh in mIMCD cells treated with one of two unrelated shRNAs targeting Bdh2 or a control enhanced green fluorescent protein (EGFP), luciferase (Luc) or non-silencing (NS) shRNA. The results confirm that the two Bdh2 shRNAs efficiently knock down Bdh2 expression in mIMCD cells.

(B-E) Cytoplasmic iron measurements. mIMCD cells expressing a Bdh2 or control shRNA were analyzed for total cytoplasmic iron levels by colorimetric analysis (B), for free cytoplasmic iron concentration by a fluorescence calcein assay (C), for ferritin-L levels by immunoblot analysis (D), and for ROS by fluorometric analysis of CDCF-DA fluorescence (E). Error bars indicate SD. The results demonstrate, consistent with our findings in FL5.12 cells, that cytoplasmic iron levels are elevated in BDH2 KD mIMCD cells.

(F-J) Mitochondrial iron measurements. mIMCD cells expressing a Bdh2 or control shRNA were analyzed for total mitochondrial iron levels by colorimetric analysis (F), for RPA (G) and RPAC (H) fluorescence by fluorometry, for DHR 123 oxidation in the presence or absence of H₂O₂ by flow cytometry (I), and for mitochondrial aconitase levels (J). Error bars indicate SD. The results demonstrate, consistent with our findings in FL5.12 cells, that mitochondrial iron levels are decreased in BDH2 KD mIMCD cells.