GAS6 is a p53 target in HPCs. (A) p53 wild-type (wt) or knockout (KO) K562 cells were treated with hemin to induce erythroid differentiation for 4 days, at which time chromatin immunoprecipitation for p53 was performed, with peaks at the human GAS6 locus shown. (B) FANCA-expressing or deficient HPCs generated by EHT in the presence of absence of doxycycline, respectively, were collected, and expression of GAS6 was analyzed by quantitative PCR (n = 6 biologic replicates results). (C) Conditioned culture supernatant of day 8 EHT cells was used for ELISA measurement of GAS6 protein (n = 13 biologic replicates compiled over 2 independent ELISA experiments). (D) FANCA-expressing HPCs were treated with or without nutlin-3a and expression of GAS6 measured by quantitative PCR (n = 3 biologic replicates results presented as mean ± SEM and compared by a Student t test with P value shown). (E) The indicated cells were treated either with BMS-777607 or GAS6 during EHT and then used in a colony formation assay (n = 7 biologic replicates over 3 experiments). (F) p53-repaired K562 cells were cultured serum free for 24 hours during which time they were treated with GAS6, and protein was collected for western blot analysis. (G) FANCA-deficient HPCs generated in the absence of doxycycline either in the presence of absence of recombinant GAS6 were harvested at day 5 of EHT and plated in methylcellulose medium, where colony formation was quantified after 14 days (n = 9 biologic replicates including 3 cell lines over 5 independent experiments). (H) Day 8 EHT cells generated without doxycycline and with or without GAS6 were exposed to mitomycin C and γH2AX-positive foci per cell quantified (n = 3 cell lines included and pooled in the overall analysis). (I) Primary FA patient bone marrow mononuclear cells were treated with or without GAS6 for either 3 or 7 days and then used in a colony formation assay, where colonies were quantified at day 14. All results presented as mean ± SEM and compared by a Student t test with P value shown.