atf4 antibody 11815  (Cell Signaling Technology Inc)

Journal: iScience

Article Title: MSMO1 promotes chemotherapy resistance through modulation of T-MAS metabolism via PERK/elF2α/ATF4/CHOP pathway

doi: 10.1016/j.isci.2026.114790

Figure Lengend Snippet: MSMO1 inhibits cell death mediated by the PERK/eIF2α/ATF4/CHOP pathway (A) Gene set enrichment analysis revealing the pathways significantly changed in MSMO1-KD cells and the control cells. (B) Gene set enrichment analysis revealing the pathways significantly changed in MSMO1-KD cells and the control cells with the pretreatment of 100 nM Tg for 3 h. (C and D) Western blotting analysis showing the expression of BIP, PERK, p -eIF2α, ATF4, CHOP in MSMO1-KD cells and overexpression cells and the corresponding control cells with or without the pretreatment of 100 nM thapsigargin (Tg) for 3 h. (E) Representative electron microscope images of untreated and Tm-treated (1 μg/mL, 24 h) MSMO1-KD and control cells. Arrowheads indicate the ER. 133 ER widths were measured in each group. x5k scale bars, 5 μm. x25k scale bars, 1 μm. (F) Cell apoptosis analysis of SK-BR-3 MSMO1 overexpressing cells and control cells treated with 100 nM Tg for 48 h. (G) Cell apoptosis analysis of MDA-MB-231 MSMO1-KD cells and control cells treated with 100 nM Tg for 48 h. (H and I) IC50 curves of MSMO1-overexpressing and MSMO1-KD cells, along with the corresponding control cells, treated with Tg, with or without pretreatment with 1 μM GSK2656157 (PERKi) for 3 h. Data are represented as mean ± SEM. (J) Representative images of thioflavin T (ThT) staining assay in MSMO1 overexpressing and control cells with or without the pretreatment of 100 nM Tg for 3 h. Scale bars, 10 μm ∗ p < 0.05; ∗∗∗∗ p < 0.0001.

Article Snippet: Rabbit monoclonal ATF4 antibody , Cell Signaling Technology , Cat# 11815; RRID: AB_2616025.

Techniques: Control, Western Blot, Expressing, Over Expression, Microscopy, Staining

Journal: iScience

Article Title: MSMO1 promotes chemotherapy resistance through modulation of T-MAS metabolism via PERK/elF2α/ATF4/CHOP pathway

doi: 10.1016/j.isci.2026.114790

Figure Lengend Snippet: MSMO1 modulates the relative content of T-MAS to regulate endoplasmic reticulum stress (A) Schematic diagram of cholesterol metabolic pathways, starting with squalene. (B) Heatmap showing the cholesterol metabolites with the most statistically significant differences between MSMO1-KD MDA-MB-231 and control cells. (C) Volcano plots showing differential metabolites between MSMO1-KD and control cells. T-MAS exhibited a significant increase (Log 2 (fold change) = 5.66 p < 0.001) in MSMO1-KD cells. (D) Representative images of thioflavin T (ThT) staining assay with or without the treatment of 1 mM 4-PBA for 3 h and 1 μg/mL T-MAS for 48 h. Scale bars, 10 μm. (E) Western blotting analysis showing the expression of BIP, PERK, p -eIF2α, ATF4, and CHOP in MDA-MB-231 cells with or without the pretreatment of 1 μM PERKi for 3 h and the treatment of 1 μg/mL T-MAS for 48 h. (F and G) IC50 curves of MSMO1 overexpressing and MSMO1-KD cells and the corresponding control cells for Tg with or without the treatment of 1 μg/mL T-MAS. Data are represented as mean ± SEM.

Article Snippet: Rabbit monoclonal ATF4 antibody , Cell Signaling Technology , Cat# 11815; RRID: AB_2616025.

Techniques: Control, Staining, Western Blot, Expressing

Journal: iScience

Article Title: MSMO1 promotes chemotherapy resistance through modulation of T-MAS metabolism via PERK/elF2α/ATF4/CHOP pathway

doi: 10.1016/j.isci.2026.114790

Figure Lengend Snippet: T-MAS induces ER stress and activates PERK/eIF2α/ATF4/CHOP pathway (A) Diagram of the cholesterol metabolism pathway, using siRNA to inhibit TM7SF2 to reduce the cellular levels of T-MAS. (B and C) Western blotting analysis showing the expression of BIP, PERK, p -eIF2α, ATF4, CHOP in MSMO1-KD and control cells with or without siRNA interfering with TM7SF2. (D) Diagram of the cholesterol metabolism pathway, using siRNA to inhibit CYP51A1 to reduce the cellular levels of FF-MAS and downstream metabolites. (E) Western blotting analysis showing the expression of BIP, PERK, p -eIF2α, ATF4, CHOP in wild-type SK-BR-3 cells with or without the pretreatment of 100 nM Tg for 3 h and siRNA interfering with CYP51A1. (F) Western blotting images showing the expression of BIP, PERK, p -eIF2α, ATF4, and CHOP in MSMO1-KD and control MDA-MB-231 cells with or without siRNA interfering with CYP51A1. (G and I) Schematic view of silencing TM7SF2 through siRNA to inhibit the transformation of FF-MAS to T-MAS, followed by the administration of FF-MAS or T-MAS to the cells. (H and J) Western blotting analysis showing the expression of BIP, PERK, p -eIF2α, ATF4, and CHOP with or without siRNA interfering with TM7SF2 and 1 μg/mL FF-MAS(or 1 μg/mL T-MAS) treatment of 48 h.

Article Snippet: Rabbit monoclonal ATF4 antibody , Cell Signaling Technology , Cat# 11815; RRID: AB_2616025.

Techniques: Western Blot, Expressing, Control, Transformation Assay

Journal: Scientific Reports

Article Title: ATF4 regulates mitochondrial dysfunction and mitophagy, contributing to corneal endothelial apoptosis

doi: 10.1038/s41598-026-36453-x

Figure Lengend Snippet: Differential activation of ER stress markers after chronic ER stress in normal human corneal endothelium cell line (21T), Fuchs (F35T) cell line, primary human corneal endothelial cells, and human corneal tissues. (A) Representative western blot data showing differential activation of ER stress-related proteins (p-eIF2α, eIF2α, CHOP, and ATF4) in 21T and F35T cell lines under DMSO (0.2%) and Tun (10 µg/mL) for 24 h; DMSO was used as solvent control. (B) Quantification of western blots demonstrating increased expression of p-eIF2α, ATF4 and CHOP in F35T compared to 21T after Tun ( n = 3–5, * P <0.05, ** P < 0.01, *** P < 0.001, one-way ANOVA with Tukey’s multiple comparison test). (C) Immunostaining images ATF4 (green) and mitochondria (labeled with mitotracker red) in F35T and 21T cell lines under normal physiological conditions. (D) Quantification of immunostaining data showing increased % of cells with ATF4 induction in F35T compared to 21T cell line under normal physiological conditions ( n = 3, ** P < 0.01, unpaired t-test). (E) Immunostaining images showing ATF4 activation after treatment with Tun (10 µg/mL, 24 h) in primary human corneal endothelial cells. (F) Immunostaining showing ATF4 and CHOP induction after treatment with Tun (10 µg/mL, 24 h) in human corneal endothelial tissues. (scale bar: 40 μm for all immuno images).

Article Snippet: For ATF4/Mitotracker staining, the cells were incubated with MitoTracker Deep Red FM (50 nM) (cat no. M22426 ; Invitrogen) for 30 min, then fixed with paraformaldehyde (PFA) (4%) (cat no. J19943.K2; Thermo Fisher) for 20 min, blocked for one hour in blocking buffer (5% normal goat serum + 0.3% Triton X-100 [cat no. X100; Millipore Sigma]) and then incubated with rabbit anti- ATF4 antibody (cat no. 11815 S, 1:1000; Cell Signaling, Danvers, MA, USA) overnight at 4°C.

Techniques: Activation Assay, Western Blot, Solvent, Control, Expressing, Comparison, Immunostaining, Labeling

Journal: Scientific Reports

Article Title: ATF4 regulates mitochondrial dysfunction and mitophagy, contributing to corneal endothelial apoptosis

doi: 10.1038/s41598-026-36453-x

Figure Lengend Snippet: ATF4 knockdown attenuates ER stress, MMP loss, mitophagy-mediator proteins, mitochondrial fragmentation, and apoptosis under chronic ER stress. (A) Western blots showing upregulation of pro-apoptotic ER stress proteins (ATF4, CHOP), downregulation of mitochondria mass related proteins (Mfn2, Tim23), upregulation of apoptotic proteins (cleaved caspase 3, cleaved caspase 9) and differential regulation of mitophagy mediator proteins (increaed Parkin and decreased PINK1) in Tun-treated 21T cell line groups (10 µg/mL, 24 h) compared to DMSO (0.2%, 24 h) under control siRNA and reversal of these ER, mitochondrial, mitophagy as well apoptosis proteins expression in ATF4-knockdown groups, compared to control siRNA under Tun. (B) Bar graph demonstrating MMP loss post Tun treatment (10 µg/mL Tun) comapred to DMSO (0.2%) under control siRNA at 48 h in primary corneal endothelial cells, while ATF4 knockdown rescues MMP loss compared to control siRNA under Tun. (C) Bar graph demonstrating loss of cell viability using MTT post Tun treatment (10 µg/mL Tun) compared to DMSO (0.2%) under control siRNA and rescue of cell death post ATF4 siRNA treatment compared to control siRNA under Tun in primary corneal endothelial cells at 24 h (for MMP and MTT, n = 3–4, * P < 0.05, *** P < 0.001, one-way ANOVA with Tukey’s multiple comparison test). (D) Immunostaining for ATF4 (green) and cytochrome c (red), DAPI (blue) in primary human corneal endothelial cells for control and ATF4 siRNA under DMSO (0.2%) and Tun (10 µg/mL) at 24 h (scale bar: 40 μm). (E) Quantification of immunostaining data showing a significant increase in % of cells with fragmented mitochondria and mitochondrial fragmentation count post Tun treatment compared to DMSO under control siRNA and attenuation of mitochondrial fragmentation after ATF4 siRNA compared to control siRNA under Tun (10 µg/mL) ( n = 5, * P < 0.05, ** P < 0.01, one-way ANOVA with Tukey’s multiple comparison test).

Article Snippet: For ATF4/Mitotracker staining, the cells were incubated with MitoTracker Deep Red FM (50 nM) (cat no. M22426 ; Invitrogen) for 30 min, then fixed with paraformaldehyde (PFA) (4%) (cat no. J19943.K2; Thermo Fisher) for 20 min, blocked for one hour in blocking buffer (5% normal goat serum + 0.3% Triton X-100 [cat no. X100; Millipore Sigma]) and then incubated with rabbit anti- ATF4 antibody (cat no. 11815 S, 1:1000; Cell Signaling, Danvers, MA, USA) overnight at 4°C.

Techniques: Knockdown, Western Blot, Control, Expressing, Comparison, Immunostaining

Journal: Scientific Reports

Article Title: ATF4 regulates mitochondrial dysfunction and mitophagy, contributing to corneal endothelial apoptosis

doi: 10.1038/s41598-026-36453-x

Figure Lengend Snippet: ATF4 knockdown decreases parkin-mediated mitophagy mediator proteins; however, it activates mitophagy in response to chronic ER stress. (A) Western blot data from the isolated mitochondrial fraction of 21T cells showing increased expression of ATF4, differential activation of mitophagy mediators (increased Parkin and LC3II and decreased PINK1) post ER stress (0.1 µg/mL Tun at 24 h) under control siRNA, and reversal of expression of mitophagy mediators under ATF4 siRNA compared to control siRNA post Tun. (B) TEM images showing mitochondria (denoted by &) and mitophagosomes (denoted by #) under control siRNA and ATF4 siRNA with DMSO and Tun treatment (10 µg/mL). (C) Quantitive bar graph showing increased number of mitophagsomes in Tun condition compared to DMSO under control siRNA and a significant decrease in number of mitophagosomes under ATF4 siRNA groups compared to control siRNA post Tun (a total of 20–25 cells have been imaged using TEM for counting mitophagosomes, scale bar: 1 μm).

Article Snippet: For ATF4/Mitotracker staining, the cells were incubated with MitoTracker Deep Red FM (50 nM) (cat no. M22426 ; Invitrogen) for 30 min, then fixed with paraformaldehyde (PFA) (4%) (cat no. J19943.K2; Thermo Fisher) for 20 min, blocked for one hour in blocking buffer (5% normal goat serum + 0.3% Triton X-100 [cat no. X100; Millipore Sigma]) and then incubated with rabbit anti- ATF4 antibody (cat no. 11815 S, 1:1000; Cell Signaling, Danvers, MA, USA) overnight at 4°C.

Techniques: Knockdown, Western Blot, Isolation, Expressing, Activation Assay, Control

Journal: Scientific Reports

Article Title: ATF4 regulates mitochondrial dysfunction and mitophagy, contributing to corneal endothelial apoptosis

doi: 10.1038/s41598-026-36453-x

Figure Lengend Snippet: Genetic deletion of ATF4 decreases pro-apoptotic ER stress molecule, CHOP, and protects against UVA-induced cell death. (A) Representative Zonula occludens-1 (ZO-1, green) immunostaining showing enlargement and loss of corneal endothelial cells (CEnCs) in ATF4 +/+ compared to ATF4 +/− mice at week 4 post-UVA (200 J/cm 2 ). (B) Bar graph representing a significant decrease in CEnC count in ATF4 +/+ compared to ATF4 +/− mice post-UVA. (C) Representative immunostained images for CHOP (green) in ATF4 +/+ and ATF4 +/− mice CEnCs at day 1 post-UVA. (D) Bar graph demonstrating a significant increase in % cells with CHOP in ATF4 +/+ compared to ATF4 +/− mice post-UVA ( n = 5, ** P < 0.01, *** P < 0.001, **** P < 0.0001 one-way ANOVA with Tukey’s multiple comparison test) (UVA: 200 J/cm 2 , Scale bar: 40 μm).

Article Snippet: For ATF4/Mitotracker staining, the cells were incubated with MitoTracker Deep Red FM (50 nM) (cat no. M22426 ; Invitrogen) for 30 min, then fixed with paraformaldehyde (PFA) (4%) (cat no. J19943.K2; Thermo Fisher) for 20 min, blocked for one hour in blocking buffer (5% normal goat serum + 0.3% Triton X-100 [cat no. X100; Millipore Sigma]) and then incubated with rabbit anti- ATF4 antibody (cat no. 11815 S, 1:1000; Cell Signaling, Danvers, MA, USA) overnight at 4°C.

Techniques: Immunostaining, Comparison

Journal: iScience

Article Title: Basal level of ATF4 promotes T cell readiness for activation-induced proliferation

doi: 10.1016/j.isci.2025.114277

Figure Lengend Snippet: ATF4 protein is upregulated in CD8 + T cells at 12 h post-activation, and Atf4 deficiency results in impaired T cell activation within 24 h (A) Quantitative RT-PCR analysis of Atf4 transcripts in CD8 + T cells at the indicated time points before and after activation (left) and Integrative Genomics Viewer (IGV) analysis of ATAC-seq coverage of ATF4 obtained from unstimulated human T cells ( GSE187659 ). CD44 lo CD62L hi naive CD8 + T cells purified from wild type (WT) mice were stimulated with anti-CD3 and anti-CD28 antibodies in the complete RPMI1640 media with 10% fetal calf serum. β-actin was used as the housekeeping gene control. N = 2–5. (B) Western blotting of ATF4 and related signaling molecules in purified naive CD8 + T cells upon stimulation with anti-CD3 and anti-CD28 antibodies. The numbers below the bands represent the signal intensity of the bands. (C) Immunoblot analysis of ATF4, GCN2, and p70S6K in CD8 + T cells after 12 h of activation in the presence or absence of mTOR inhibitors (rapamycin (20 nM) and Torin 1 (0.5 μM)) and GCN2 inhibitor (GCN2iB, HY-112654, 0.5 μM). The freshly purified naive CD8 + T cells were used as 0-h control. Gray values of the indicated proteins were determined for statistical analysis on the right. N = 2–4. (D) Immunoblot analysis of ATF4, GCN2, and PERK in CD8 + T cells after 12 h of activation in the presence or absence of mTOR inhibitor (rapamycin), GCN2 inhibitor (GCN2iB), and PERK inhibitor (GSK2606414, 1 μM). (E and F) Immunoblot analysis of ATF4, p70S6K, PERK, and eIF2α in CD8 + T cells after 12 h of activation in the presence of various pharmacological agents. The addition of Torin1 (0.5 μM), rapamycin (20 nM), ISRIB (0.2 μM), and Tg (0.2 μM) was at the beginning of T cell activation in (E). The addition of ISRB (0.2 μM), CHX (5 μg/mL) was at 4 h and that of CQ (20 μM) was at 8 h after T cell activation in (F). (G) Flow cytometry analysis of forward scatter (FSC), side scatter (SSC), and the percentages of CD69 + , CD98 + , and CD25 + T cells after 24 h of activation. CD8 + CD44 lo CD62L hi T cells from WT and Atf4 cKO mice were purified by flow cytometry, stimulated by anti-CD3 and anti-CD28 antibodies, and subjected to flow cytometry analysis. The geometric mean of fluorescence intensities of MitoSOX Red (mitochondrial ROS), mitochondrial membrane potential (MitoSpy), DCFDA (ROS), and 2-NBDG (glucose uptake) staining of T cells at 24 h post-activation is also shown. (H) Western blotting of the phosphorylation of p70S6K and GCN2 (24 h, left) and puromycin incorporation (12 and 24 h, right) in WT and Atf4 −/− CD8 + T cells. (I) Flow cytometry analysis of EDU and 7-AAD staining of T cells at 24 h post-activation. The percentages of EDU - 7-AAD - (G0/G1 phase), EDU - 7-AAD + (G2/M phase), and EDU + (S phase) cells in WT and Atf4 −/− T cells were compared on the right. Data are representative of 2 experiments for (A-B, D, E-F, H) and 3–4 independent experiments for (C, G, I). Student’s t test was used for statistical analysis. Mean ± SD, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.005, ∗∗∗∗ p < 0.001, ns, not significant.

Article Snippet: ATF4 antibody , Cell Signaling , Cat#11815S.

Techniques: Activation Assay, Quantitative RT-PCR, Purification, Control, Western Blot, Flow Cytometry, Fluorescence, Membrane, Staining, Phospho-proteomics

Journal: iScience

Article Title: Basal level of ATF4 promotes T cell readiness for activation-induced proliferation

doi: 10.1016/j.isci.2025.114277

Figure Lengend Snippet: ATF4 transcription factor activities in CD8 + T cells early after activation CD8 + CD44 lo CD62L hi T cells were purified and stimulated by anti-CD3 and anti-CD28 antibodies for 12 and 24 h. The cells were harvested for ATF4-specific CUT&RUN analysis (12 h post-activation, A-D) and RNA-sequencing analysis (0, 12, 24 h post-activation, E-F, H). (A) Heatmap of normalized CUT&RUN signal of ATF4 and control IgG after 12 h of T cell activation. The pie chart on the right displays the location features of ATF binding sites. (B) DNA-binding motifs of ATF4 were identified by HOMER and MEME. The enriched motifs were shown. (C) Representative examples of profiles of known ATF4-regulated genes Slc7a11 , Shb , and unknown ATF4-regulated genes Map4k3 and Ccr7 for CUT&RUN data. The IgG control peaks are shown in gray, and ATF4-bound peaks are shown in blue. Ticks mark the location of significant binding regions. The Y axis was autoscaled within each region by IGV. (D) GO enrichment analysis for genes bound by ATF4. https://metascape.org . (E) KEGG analysis of genes upregulated or downregulated at indicated time points (RNA-seq). (F) Heatmap shows the expression of amino acid metabolism-associated genes in CD8 + T cells at the indicated time points. The genes were selected based on those reported to be ATF4-dependent, insulin/mTORC1-sensitive, but cellular stress-independent in mouse embryonic fibroblasts. (G) Venn diagram summarizes the number of differentially expressed genes (DEGs) at the indicated time points that were also identified by CUT&RUN at 12 h post-activation. The DEGs were obtained from RNA-seq comparison between 12-h and 0-h, or 24-h and 0-h samples. (H) Heatmap shows the expression of ATF4-bound genes (identified by CUT&RUN) in CD8 + T cells at indicated time points.

Article Snippet: ATF4 antibody , Cell Signaling , Cat#11815S.

Techniques: Activation Assay, Purification, RNA Sequencing, Control, Binding Assay, Expressing, Comparison

Journal: iScience

Article Title: Basal level of ATF4 promotes T cell readiness for activation-induced proliferation

doi: 10.1016/j.isci.2025.114277

Figure Lengend Snippet: Atf4 -deficient CD8 + T cells display altered transcriptome and metabolites in early-phase T cell activation (A) Volcano plots show the differentially expressed genes between WT and Atf4 −/− CD8 + T cells at 12- (upper panel) and 24-h (lower panel) post activation. (B) GSEA analysis of differential pathways between Atf4 −/− and WT CD8 + T cells at 12- and 24-h post-activation. NES represents the normalized enrichment score. (C) Heatmap shows the expression of amino acid metabolism-associated genes in CD8 + T cells at the indicated time points. (D) PCA analysis of the targeted metabolomic profiles of WT and Atf4 −/− T cells after 12 h of activation (left). Heatmap shows the quantification of amino acids between WT and Atf4 −/− T cells after 12 h of activation (right). (E) Comparison of reduced-to-oxidized glutathione ratio between WT and Atf4 −/− T cells after 12 h of activation. student's t test was used for statistical analysis. Mean ± SD, ∗p < 0.05. (F) GSEA plots of regulation of response to oxidative stress, PERK-mediated UPR, and intrinsic apoptotic signaling pathway in response to ER stress for the comparison of the transcriptome of Atf4 −/− and WT T cells after 12 h of activation.

Article Snippet: ATF4 antibody , Cell Signaling , Cat#11815S.

Techniques: Activation Assay, Expressing, Metabolomic, Comparison

Journal: iScience

Article Title: Basal level of ATF4 promotes T cell readiness for activation-induced proliferation

doi: 10.1016/j.isci.2025.114277

Figure Lengend Snippet: Atf4 -defiient CD8 + T cells exhibit altered transcriptome kinetics CD8 + CD44 lo CD62L hi T cells from WT and Atf4 cKO mice were purified by flow cytometry, stimulated by anti-CD3 and anti-CD28 antibodies, and subjected to RNA-seq analysis. (A) PCA analysis of the transcriptome of WT (triangle) and Atf4 −/− (circle) CD8 + T cells at the indicated time points (indicated by graded red). (B) Relationships between clusters identified in WT (W1-W5) and Atf4 −/− (K1-K5) groups by fuzzy c-means clustering. Arrows indicate shared genes between clusters, showing the overlap and correspondence between WT and Atf4 −/− cells. (C) Time-series gene expression dynamics for clusters identified in WT (W1-W5) and Atf4 −/− (K1-K5) groups. The heatmap shows the temporal expression patterns (0 h, 12 h, and 24 h) for genes in each cluster. Functional annotations on the right highlight biological processes and pathways enriched in each cluster. (D) GO enrichment analysis of common genes between clusters W1 and K3 (upper left panel), W4 and K3 (lower left panel), W2 and K4 (upper right panel), W5 and K5 (lower right panel). https://metascape.org .

Article Snippet: ATF4 antibody , Cell Signaling , Cat#11815S.

Techniques: Purification, Flow Cytometry, RNA Sequencing, Gene Expression, Expressing, Functional Assay

Journal: iScience

Article Title: Basal level of ATF4 promotes T cell readiness for activation-induced proliferation

doi: 10.1016/j.isci.2025.114277

Figure Lengend Snippet: CD8 + T cells deficient in Atf4 exhibit reduced chromatin accessibility during the early stage of T cell activation CD8 + CD44 lo CD62L hi T cells from Atf4 cKO and WT mice were purified by flow cytometry and were stimulated by anti-CD3 and anti-CD28 antibodies for 12 h. The cells were then subjected to ATAC-sequencing. (A) Genome-wide comparison of chromatin accessibilities (peaks) identified by ATAC-seq in WT and Atf4 −/− CD8 + T cells after 12 h of activation. Downregulated peaks in Atf4 −/− CD8 + T cells are shown on the left while upregulated peaks in Atf4 −/− CD8 + T cells are shown on the right. (B) Pearson’s correlation of the 2 WT and 2 cKO samples (upper panel) and the pie chart (lower panel) displaying the location features of downregulated peaks identified by ATAC-seq. (C) GSEA analysis of genes with downregulated (upper panel) and upregulated (lower panel) peaks in Atf4 −/− compared to WT T cells. (D) Representative examples of profiles of Tbx21 , Tbk1 , Map2k3 , Tcf7 , and Lag3 for ATAC-seq data. The peaks from WT cells are shown in gray and those from Atf4 −/− are shown in blue. Ticks mark the location of differential peaks. The Y axis was autoscaled within each region by IGV. (E) Transcription factor motif variability in chromatin accessibility between WT and Atf4 −/− CD8 + T cells. Upper panel: Variability scores of transcription factor motifs. Top motifs, such as Stat5a:Stat5b, FOS::JUN, Stat4, and Bcl6, show the highest variability in accessibility. Lower panel: Heatmap shows the z-scores of each bias-corrected deviation in accessibility for selected transcription factor motifs. Positive deviations indicate increased motif accessibility, while negative deviations indicate decreased motif accessibility. (F) Venn plot on the left shows the number of common peaks identified in CUT&RUN sequencing of WT T cells and differentially regulated in ATAC-seq of Atf4 −/− T cells at 12 h post-activation. Heatmap in the middle shows the chromatin accessibility of the downregulated peaks in CD8 + WT and Atf4 −/− T cells after 12 h of activation. Heatmap on the right showing the interaction intensity of ATF4-bound genes with downregulated chromatin accessibility in CD8 + WT T cells after 12 h of activation. (G) GO enrichment analysis of the common genes identified in CUT&RUN sequencing of WT T cells and downregulated in the ATAC sequencing of Atf4 −/− T cells at 12 h post-activation. (H) Venn plot shows the number of common genes downregulated in ATAC-seq and RNA-seq of Atf4 −/− T cells at 12 h post-activation. (I) GO enrichment analysis of the common genes downregulated in ATAC-seq and RNA-seq of Atf4 −/− T cells at 12 h post-activation.

Article Snippet: ATF4 antibody , Cell Signaling , Cat#11815S.

Techniques: Activation Assay, Purification, Flow Cytometry, Sequencing, Genome Wide, Comparison, RNA Sequencing

Journal: iScience

Article Title: Basal level of ATF4 promotes T cell readiness for activation-induced proliferation

doi: 10.1016/j.isci.2025.114277

Figure Lengend Snippet: ATF4-regulated genes in CD8 + T cells at 48 h post-activation (A-F) and defects in Atf4 −/− CD8 + T cells at 72 h post-activation (G-I). CD8 + CD44 lo CD62L hi T cells from WT and Atf4 cKO mice were purified by flow cytometry and were stimulated by anti-CD3 and anti-CD28 antibodies for 48 h. WT cells were then subjected to CUT&RUN (A-C, F). WT and Atf4 −/− T cells were subjected to RNA-sequencing (D-F) and flow cytometry analysis (G-I). (A) Genome-wide comparison of ATF4 binding in CD8 + T cells. Heatmaps are shown on the left. The pie chart on the right displays the location features of ATF binding sites. (B) DNA-binding motifs of ATF4 were identified by HOMER. The enriched motifs were shown. (C) GO enrichment analysis of the genes identified in the CUT&RUN sequencing of WT T cells at 48 h post-activation is shown on the lower left. Representative examples showing ATF4-regulated genes for CUT&RUN data are shown on the right. The IgG control peaks are shown in gray, and ATF4-bound peaks are shown in blue. Ticks mark the location of significant binding regions. The Y axis is autoscaled within each region by IGV. (D) RNA-seq comparison of WT and Atf4 −/− CD8 + T cells at 48 h post-activation. Left panel, volcano plot showing the differentially expressed genes between cKO and WT T cells. Right panel, GSEA analysis of DEGs between Atf4 −/− and WT CD8 + T cells after 48 h of stimulation. (E) Heatmaps compare the expression of leading-edge genes in “intrinsic apoptotic signaling pathway in response to ER stress” (T cell transcriptome at 12 h post-activation) and in “PERK regulates gene expression” (T cell transcriptome at 48 h post-activation). (F) Common genes identified by CUT&RUN analysis in WT cells and differentially expressed genes identified between Atf4 −/− and WT CD8 + T cells at 48 h post-activation are shown as a Venn plot on the left (gene numbers) and heatmap on the right panel (gene expression). (G) Flow cytometry comparison of cell proliferation between Atf4 −/− and WT CD8 + T cells at 72 h post-activation. The results of Ki67 and CD44 staining are shown on the left panel, and those of CFSE dilution are shown on the right. (H) Flow cytometry analysis of the percentages of T-bet + and CD44 + cells in Atf4 −/− and WT CD8 + T cells at 72 h post-activation. (I) Intracellular staining of TNF-α and IFN-γ in Atf4 −/− and WT CD8 + T cells after 72 h of activation by anti-CD3 and anti-CD28 antibodies and 4 h of restimulation by PMA and ionomycin. The percentages of IFN-γ + , TNF-α + , and IFN-γ + TNF-α + cells are shown. The experiments in (G-I) were repeated for 2–3 times. Student’s t test was used for statistical analysis. Mean ± SD, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.005, ns, not significant.

Article Snippet: ATF4 antibody , Cell Signaling , Cat#11815S.

Techniques: Activation Assay, Purification, Flow Cytometry, RNA Sequencing, Genome Wide, Comparison, Binding Assay, Sequencing, Control, Expressing, Gene Expression, Staining

Journal: iScience

Article Title: Basal level of ATF4 promotes T cell readiness for activation-induced proliferation

doi: 10.1016/j.isci.2025.114277

Figure Lengend Snippet: Atf4 −/− CD8 + T cells have impaired antiviral responses (A) Transcriptome comparison of Atf4 expression in P14 T cells at 7 days post-LCMV infection. The bulk RNA sequencing data of P14 transgenic T cells at 7 days after LCMV Armstrong or CL13 infection, with the accession number GSE11943 , were analyzed. (B–E) WT and cKO mice were infected with LCMV CL13 at 4 × 10 5 PFU via intraperitoneal injection and were analyzed at 10 dpi. (B) Quantitative RT-PCR of LCMV-glycoprotein (GP) mRNA in peripheral blood and indicated tissues at 10 days post-infection (dpi). The mRNA series of dilutions of stock LCMV with known viral titers was used as a standard curve for calculating the viral titer in peripheral blood samples. N = 2–3. (C) Flow cytometry analysis of the percentages and cell numbers of total CD8 + T cells and CX3CR1 + SLAMF6 - CD8 + T cells in the spleen, liver, and lung of WT and cKO mice. N = 4. (D) Flow cytometry analysis of T-bet and Ki67 expression in CD8 + T cells. N = 4. (E) Flow cytometry analysis of IFN-γ and TNF-α expression in CD8 + T cells. N = 5. Data are representative of 3–4 independent experiments for (B-E). Student’s t test was used for statistical analysis. Mean ± SD, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.005, ∗∗∗∗ p < 0.001, ns, not significant.

Article Snippet: ATF4 antibody , Cell Signaling , Cat#11815S.

Techniques: Comparison, Expressing, Infection, RNA Sequencing, Transgenic Assay, Injection, Quantitative RT-PCR, Flow Cytometry