Skip to main content
. Author manuscript; available in PMC: 2018 May 30.
Published in final edited form as: Nature. 2017 Nov 22;551(7682):639–643. doi: 10.1038/nature24637

Extended Data Figure 2. Core components of the ISC machinery exhibit differential requirements, dependent on O2 concentration.

Extended Data Figure 2

a, Five-day proliferation assays of the indicated cell lines upon introduction of shRFP (black) or shRNAs targeting NFS1 (shNFS1_1 or shNFS1_2, grey) at the indicated O2 concentrations. Data are from three biological replicates. Significant P values (asterisks) are as follows: MCF10DCIS.com bar 1 versus bar 2, 5 × 10−6; bar 1 versus bar 3, 9 × 10−7; bar 2 versus bar 5, 5 × 10−6; bar 3 versus bar 6, 2 × 10−6; bar 5 versus bar 8, 2 × 10−6; bar 6 versus bar 9, 3 × 10−6; MDA-MB-231 (right) bar 1 versus bar 2, 5 × 10−8; bar 1 versus bar 3, 3 × 10−8; bar 2 versus bar 5, 1 × 10−7; bar 3 versus bar 6, 9 × 10−5. b, Weight or volume of orthotopic tumour xenografts derived from MCF10DCIS.com cells expressing shRFP, shNFS1_1, or shRNA targeting ABCB7 (shABCB7_2), 4 weeks of growth. Data are from four independent biological replicates. ns, not significant. c, Five-day proliferation assays of MCF10DCIS.com cells expressing shRFP (black) or shNFS1_1 (grey) (top). Data are from three independent biological triplicates. Immunoblots of NFS1 and RPS6 (bottom). Images are representative of three experiments. Significant P values (asterisks) are as follows: 6% O2, 2 × 10−2; 10% O2, 4 × 10−5; 14.5% O2, 3 × 10−4; 21% O2, 5 × 10−6. d, Five-day proliferation assays at atmospheric O2, MCF10DCIS.com cells stably transduced with vector control (VC), NFS1 cDNA (NFS1res), or catalytically inactive mutant cDNA (NFS1resCD) (left). Immunoblots of NFS1 and RPS6 upon introduction of shRFP (R) or shNFS1_1 (N1), right. Data are from three independent biological replicates. e, Five-day proliferation assays as in a upon introduction of shRFP or shRNAs targeting ABCB7 (shABCB7_1, shABCB7_2) (left, centre).* , Significant P values: MCF10DCIS.com bar 1 versus bar 2, 1 × 10−7; bar 1 versus bar 3, 5 × 10−7; bar 2 versus bar 5, 4 × 10−7; bar 3 versus bar 6, 2 × 10−7; MDA-MB-231 bar 1 versus bar 2, 3 × 10−6; bar 1 versus bar 3, 3 × 10−6; bar 2 versus bar 5, 7 × 10−6; bar 3 versus bar 6, 1 × 10−6. ABCB7 mRNA levels normalized to ACTB mRNA and relative to shRFP, (right). Data are from three independent biological replicates. f, Five-day proliferation assays of MCF10DCIS.com cells upon introduction of shRFP (black) or shRNAs targeting FXN (shFXN_1 or shFXN_2, grey) or ISCU (shISCU, blue) at the indicated O2 concentrations or upon addition of the antioxidant trolox (100 µM). Data are from three independent biological triplicates (left). FXN or ISCU mRNA levels normalized to ACTB mRNA and relative to shRFP (right). g, Proliferation assay (left, data from three independent biological triplicates) and mRNA levels (right, data from three independent biological replicates) as in d upon introduction of RFP (shRFP, black), or shRNAs targeting MOCS3 (shMOCS3_1 or shMOCS3_2). MOCS3 is an enzyme essential for molybdenum co-factor biosynthesis, a process in which NFS1 has been proposed to play a critical role independent of ISC biosynthesis10. a–g Data are mean ± s.e.m., P values obtained by heteroscedastic two-sided t-test.