Fig 2: ATF3 binds to the enhancer/promoter regions of SSP genes(A–C) Genome Browser views of ATF3-binding peaks at regions proximal to transcription start sites of PHGDH (A), PSAT1 (B), or PSPH (C). The ChIP-seq data were generated using ATF3-WT and KO HCT116 cells. Data from WT cells treated with camptothecin (CPT, to induce ATF3 expression) were also presented if basal ATF3 binding was not present or too weak. ATF4 peaks were derived from the ENCODE database, while the enrichment of H3K27ac was also shown.(D–G) RPE cells cultured in the serine/glycine-deprived medium for 0, 4, 8, and 24 h were subjected to ChIP assays using immunoglobulin G (IgG) or the ATF3 antibody. The amounts of precipitated DNA were quantified by qPCR using primers amplifying the regions indicated in (A), (B), or (C) or a non-specific (NS) region indicated in Figure S5A. The data are presented as mean ± SD (n = 3).(H) ATF3-KO RPE cells were serine starved for 8 h and subjected to ChIP assays using IgG or the ATF3 antibody. The specific PHGDH and NS region were amplified and quantitated by qPCR.(I) Cells were co-transfected with a minimal reporter (MiniP), or the PHGDH-enhancer-reporter (en-MiniP), with or without ATF3 or ATF4 for dual luciferase assays. The enhancer-reporter construct was depicted above the plot. n = 3.(J and K) ATF4-WT (ATF4+/+) and KO (ATF4−/−) HEK293T cells were infected with lenti-ATF3 (ATF3) or control viruses (EV, empty vector) and subjected to serine starvation for qRT-PCR assays.The data are presented as mean ± SD (n = 3). **p < 0.01; ****p < 0.0001; Student’s t test; compared at the same time point between WT and KO cells. See also Figure S5.

Fig 3: ATF3 deficiency impairs serine-deprivation-induced expression of serine-synthesis-pathway genes(A) A diagram shows the serine synthesis pathway. Filled circles represent [13C] atoms derived from [U-13C]-D-glucose.(B) Heatmap presentation of genes regulated by ATF3 upon serine deprivation. WT or ATF3-KO RPE cells cultured in the complete or the serine/glycine-depleted medium for 8 h (2 biological repeats for each sample) were subjected to RNA-seq. Gene expression changes induced by serine deprivation were used to plot the heatmap. The cutoff value (2.5) was chosen to allow for better presentation of differential expression of the majority of genes.(C) DAVID (Database for Annotation, Visulaization and Integrated Discovery) GO analysis of ATF3-regulated genes upon serine deprivation.(D) Indicated ATF3KO or control cells were cultured in the serine/glycine-deprived medium for 0, 4, 8, 24, and 48 h for qRT-PCR assays. The data are presented as mean ± SD (n = 3).(E) Indicated cells cultured in the serine/glycine-deprived medium for 0, 4, 8, 24, and 48 h were subjected to western blotting for PHGDH and PSAT1 expression. The intensities of PHGDH/PSAT1 bands are quantified using ImageJ, normalized to that of β-actin, and indicated under corresponding blots.(F) WT and ATF3-KO HCT116 cells cultured in the medium containing 25 mM of [U-13C]-D-glucose with or without serine/glycine were lysed for metabolomics analyses. L-serine derived from [U-13C]-glucose (M+3) was quantitated and plotted as ratios to unlabeled L-serine (M+0).n = 6; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; Student’s t test. See also Figure S3.

Fig 4: Oleandrin induces ICD via ER stress.MCF7, T47D, and MDA-MB-231 cells were treated with oleandrin or DMSO for 10 h. The differential of mRNA was analyzed by mRNA sequencing. A The Venn’s diagrams show the significant difference mRNA in three different pairs of cells. B The pathway analysis was performed functional enrichment of these different mRNA in GO and KEGG databases. C The heatmap was generated based on the significant different genes in ATF2 pathway. D, E The ATF3, ATF4, and CHOP (DDIT3) expression was detected by qRT-PCR and western blotting. Data are presented as mean ± standard error of the mean from three independent experiments. **p < 0.01. ole, oleandrin.

Fig 5: Β1 integrin mediates Sema7A signal to TGF-β2 via ATF3.a Cells were treated with β1 integrin antibody (P5D2), and ATF3 mRNA level was analyzed by qPCR normalized to GAPDH. Fold changes are shown. Data are mean ± SEM, N = 3, *p < 0.05; **p < 0.0.1. b ATF3 protein expression was analyzed by western blotting, normalized to tubulin. Data are mean ± SEM, N = 3, *p < 0.05; **p < 0.01. c TGF-β2 mRNA level was analyzed by qPCR normalized to GAPDH. Fold changes are shown. Data are mean ± SEM, N = 3, *p < 0.05; **p < 0.0.1. d The concentration of TGF-β2 in cell supernatant was detected by ELISA for Con335-HUVECs, Sema7A-HUVECs, and Sema7A-HUVECs + P5D2. Data are mean ± SEM, N = 10, *p < 0.05; ***p < 0.001. e, f Smad3 phosphorylation were analyzed by western blotting, normalized to total Smad3. Data are mean ± SEM, N = 3, *p < 0.05. g, h CD31 and α-SMA mRNA level were analyzed by qPCR normalized to GAPDH. Fold changes are shown. Data are mean ± SEM, N = 3, *p < 0.05; **p < 0.01; ***p < 0.001. i–k CD31 and α-SMA proteins were analyzed by western blotting, normalized to tubulin. Data are mean ± SEM, N = 3, *p < 0.05; **p < 0.01; ***p < 0.001. l ATF3 overexpression plasmid was transfected into P5D2 incubated Sema7A-HUVECs, and TGF-β2 mRNA expression in Sema7A-HUVECs + P5D2 and Sema7A-HUVECs + P5D2 + ATF3 were analyzed by qPCR normalized to GAPDH. Fold changes are shown. Data are mean ± SEM, N = 3, **p < 0.01. m–o CD31 and α-SMA protein expressions were analyzed by western blotting, normalized to GAPDH. Data are mean ± SEM, N = 3, **p < 0.01 (Sema7A-HUVECs + P5D2 vs Sema7A-HUVECs + P5D2 + ATF3). P5D2 β1 integrin antibody.