PIGA deficiency impairs hepcidin (HAMP) upregulation upon HJV overexpression. (A) Schematic representation of the PCR cassette integration in exon (Ex) 2 of PIGA. (B) mRNA levels of PIGA in PIGA WT and PIGA KO cells, normalized to parental Hep3B cells (dashed line). PIGA mRNA expression was normalized to the housekeeping gene GAPDH. (C) Western blot analysis of PIGA in parental Hep3B, PIGA WT, and PIGA KO cells. Vinculin was used as a loading control. (D) Fluorescence-activated cell sorter (FACS) analysis representing the percentage of CD59 hycoerythrin-positive cells in PIGA WT and PIGA KO clones. (E) FACS analysis of the percentage of HJV-Flag allophycocyanin-positive cells in PIGA WT and PIGA KO cells upon HJV-Flag overexpression. In panels D and E, cells were previously gated for the exclusive inclusion of singlets (FSC-A vs FSC-H) and exclusion of dead cells (7-AAD positive). (F) Hepcidin mRNA levels in PIGA WT and PIGA KO cells after overexpression of HJV, PIGA, or HJV and PIGA vectors simultaneously. EV, empty vector. (G) mRNA levels of Hepcidin in PIGA KO cells after overexpression of PIGA^WT, PIGA^L344P, PIGA^R77Q, PIGA^L110del, and PIGA^R412* mutants together with HJV. Hepcidin mRNA expression was normalized to the housekeeping gene GAPDH. (H) Western blot analysis of ceruloplasmin (CP) in PIGA WT and PIGA KO cells. Vinculin was used as a loading control. Student t test/2-way analysis of variance: */^#P < .05, **/^##P < .01, ***/^###P < .001, ****/^####P < .0001; *comparisons inside each group; ^#relative to comparisons to the corresponding WT counterpart.