Fig 1: Iron Deficiency Compromises NMD in V-ATPase-Deficient cells(A) Schematic representation of transferrin (TF)-mediated iron uptake. After receptor-mediated endocytosis via the TF receptor (TFRC), acidification of the endosome/lysosome by V-ATPase promotes iron release and transport to the cytoplasm.(B) Western blot analysis of the TFRC in EGFPPTC-231 cells after lentiviral delivery of Cas9 and NT or TFPC-targeting sgRNAs. a-Tubulin served as a loading control.(C) Flow Cytometry analysis of EGFPPTC-231 cells after lentiviral delivery of Cas9 and the indicated sgRNAs.(D and E) qRT-PCR analysis of EGFPPTC-231 (D) or EGFPPTC-35 mRNA(E) in reporter cell lines after lentiviral delivery of Cas9 and the indicated sgRNAs. All mRNA expression levels shown in this figure were first normalized to GAPDH expression and then normalized to expression level in sgNT1-infected cells.(F) Flow cytometry assay for intracellular iron content, based on calcein-AM (CA-AM) fluorescence, in control (DMSO)- or bafilomycin (5 nM)-treated HCT116 cells with or without 0.5 mM iron citrate supplementation.(G) Flow cytometry analysis of EGFPPTC-231 cells after lentiviral delivery of Cas9 and the indicated sgRNAs with or without 0.5 mM iron citrate supplementation.(H) qRT-PCR analysis of NMD targets in EGFPPTC-231 cells after lentiviral delivery of Cas9 and the indicated sgRNAs with or without 0.5 mM iron citrate supplementation.Data are represented as mean ± SD (n = 3 biological replicates for all qRT-PCR experiments). *p < 0.05, ***p < 0.001; calculated by Student’s t test. See also Figure S3.