Fig 2: Cell death is increased in Slc7a5‐null embryos, and exogenous ER stress induction in wild‐type embryos upregulates Chac1 and Trib3 A, BWestern blot of individual E9.5 wild‐type (n = 4) and Slc7a5‐null (n = 4) embryo lysates immunoblotted using an antibody against phospho‐GCN2 (Thr899) and total GCN2 (loading control), ***P = 0.0001; or against phospho‐eIF2α (Ser51) and total eIFα (loading control), *P = 0.0482, band intensities measured with FIJI and analysed with unpaired t‐test (see original source data).C–FPhosphorylated eIFα was assessed by immunofluorescence in E9.5 embryos in the forebrain, wild type (C, c1) (n = 2 embryos, 0/14 sections) and Slc7a5‐null (D, d1) (n = 2 embryos 16/16 sections), and in hindbrain/anterior spinal cord, wild type (E–e2) (n = 4 embryos, 0/62 sections) and Slc7a5‐null (F–f2) (n = 4 embryos, 54/54 sections) including in the otic vesicle (compare e2, f2). Scale bars in (C–e1, D–f1) 100 µm and in (e2 and f2) 25 µm. Arrowheads indicate ventral midline.GAn increase in Trib3 protein was detected by Western blot in Slc7a5‐null embryos; each lane represents an E9.5 embryo lysate (wild type n = 3, Slc7a5‐null, n = 4) with α‐tubulin loading control, unpaired t‐test *P = 0.0153 (see original source data).H–LTUNEL assay to detect apoptotic cells in wild type (n = 3 embryos, 32 sections) or Slc7a5‐null (n = 5 embryos, 47 sections); (H, I) transverse sections through the head at level of the forebrain and (J, K) the spinal cord, (H–K) scale bars 50 µm, arrowheads indicate ventral midline. (L) Quantification of TUNEL‐positive cells within spinal cord sections was performed as stated in Materials and Methods. Each dot represents the average apoptosis cell count for a single embryo, Welch's correction unpaired t‐test, P = 0.250, F‐test to compare variances, P = 0.0003 (see original source data).Data information: All error bars indicate SEM.Source data are available online for this figure.

Fig 3: Transcriptome profiling of wounded keratinocytes indicates altered expression of genes involved in cellular migration and the unfolded protein response.A–D, volcano plots illustrating log2fold change with p-adjusted value (−log base 10) between unwounded GCN2 KO versus WT (A), wounded GCN2 KO versus WT (B), WT wounded versus unwounded (C) and GCN2 KO wounded versus unwounded (D). E, heat map of activated molecular functions comparison between WT HDW-unwounded and GCN2 KO HDW-unwounded made using comparison tool of IPA. Heat map shows activation Z-scores with the scale showing the highest z scores in red and lowest in blue. F, table of upstream regulators of differentially expressed genes (DEG) in HDW GCN2 KO versus HDW WT group predicted using Upstream Regulator Effects Analysis tool of IPA. G, heat map of differentially expressed UPR genes and genes regulating cystine uptake identified in unwounded GCN2 KO NTERT cells. Data is depicted as log2fold change of genes in GCN2 KO unwounded versus WT unwounded group. The scale shows the highest log2fold change is denoted as red and the lowest is denoted as blue. H, heat maps illustrating a group of differentially expressed genes in wounded GCN2 KO NTERT cells. The genes are categorized based on distinct roles in different stages of cell migration. Data is represented as log2fold change of genes in GCN2 KO HDW versus WT HDW group. The scale to the right of the panels shows the highest log2fold change as denoted as red and the lowest represented as blue.

Fig 4: Proposed model of the feedback mechanism by which low amino acids suppress IL-1β expression in GPP. In GPP, IL-1β and other inflammatory cytokines induce dramatic upregulation of acute-phase proteins including SAA. SAA activates the NLRP3 inflammasome pathway and secretion of mature IL-1β from blood monocytes as an amplification loop; on the other hand, the acute-phase reaction results into amino acid starvation in circulation. Low amino acids induce activation of GCN2, which decreases ROS levels, thereby inhibiting NLRP3 inflammasome pathway and IL-1β release, as a negative feedback mechanism.

Fig 5: Model for the role the activation and function of GCN2 in KCCM and wound healing. The wound bed is illustrated, along with the leading edge of keratinocytes. Wound stress signals activation of GCN2, which is indicated by its phosphorylation. The induced GCN2 serves to maintain elevated levels of phosphorylated eIF2α and attendant translational control. With wounding of keratinocytes, there is a reduction of select free amino acids, including cysteine levels that culminate in lowered aminoacylation of tRNACys, which is suggested to be a direct activator of GCN2. Lowered cysteine levels in response to wounding are suggested to be a consequence of the demands of translation and the role of cysteine in glutathione production and ROS management. Induced GCN2 facilitates KCCM during wounding by multiple mechanisms: (1) enhanced synthesis of cystine transporters, including SLC7A11 and SLC3A2, that serves to maintain cysteine levels; (2) increased RAC-dependent ROS production by NOX enzymes; and (3) through appropriate actin remodeling. Availability of intracellular cysteine is important for glutathione production and management of ROS; and ROS generation and localization at the leading edge are important for appropriate remodeling of the cytoskeleton.