Fig 1: Loss of Abcb10 results in stress signaling through eIF2S1 phosphorylation and upregulation of ATF4 and downstream nutritional stress sensors CHOP, CHAC1 and RARS.A, RNA Seq from WT and Abcb10-null MEL cells during differentiation was performed (n = 5 biologic replicates). GSEA on reactome gene sets shows enrichment of pathways involved in stress response and amino acid metabolism at day 5. B, transcripts for eIF2S1, ATF4, downstream target of ATF4, including Chop, CHAC1, and Rars in WT and Abcb10-null MEL cells at Day 0, 2, and 5 of differentiation from (A) were expressed as FPKM. Bar graphs show the mean ± SD, n = 3–5 experiments with Student’s t test significance p = 0.05. C, Western blot analysis for eIF2S1, phospho-eIF2S1, Atf4, Chop, Chac1, Rars, and actin in WT and Abcb10-null MEL cells at Day 0, 2, and 5 of differentiation was performed as described in Experimental procedures. D, blots were quantified using Fiji Image J as described in Experimental procedures using actin as a loading control. (n = 2–3 biologic replicate/experiment; n = 3–6 experiments). The ratios of phosphorylated eIF2S1/total eIF2S1 and protein/actin are shown. Bar graphs show the mean ± SD with Student’s t test significance p = 0.05. eIF2S1, eukaryotic initiation factor-2S1; GSEA, Gene set enrichment analysis; MEL, murine erythroleukemia.