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Cell Signaling Technology Inc antibodies against atf3
Antibodies Against Atf3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 78 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/atf3+18665+antibody/ATF-3+Rabbit+mAb/pm41224102-78-10-13
Average 95 stars, based on 78 article reviews

antibodies against atf3 - by Bioz Stars, 2026-10

95/100 stars

Images

Polyacrylamide Gel Electrophoresis:

Article Title: Platelet-activating factor induces ferroptosis by binding to ATF3 and inhibiting the SLC7A11/GPX4 axis to suppress the progression of endometrial carcinoma.
Article Snippet: Following centrifugation 221 (12000 ×g, 20 min, 4 °C), the protein concentrations of the 222 supernatant were determined using a BCA Protein Assay kit (23227; 223 Thermo Fisher Scientific, USA) according to the manufacturer’s 224 protocol. .. Equal volume of protein samples were separated by SDS225 PAGE, transferred onto PVDF membranes, and then probed using 226 the following antibodies at the indicated dilutions: NRF2 (380773, 227 1:1000; Zen BioScience, China), ACSL4 (R24265, 1:1000; Zen 228 BioScience, China), SLC7A11 (382036, 1:1000; Zen BioScience, 229 China), FTH1 (381204, 1:1000; Zen BioScience, China), GPX4 (bs230 3884R, 1:1000; Biosss, China), ATF3 (18665S, 1:1000; Cell Signaling AR TIC LE IN PR ES S 231 Technology, USA), and GAPDH (5174, 1:1000; Cell Signaling 232 Technology, USA). ..

Western Blot:

Article Title: Nintedanib enhances tumor cell radiosensitivity by promoting ferroptosis and modulating the ATF4/SLC7A11/GSH axis
Article Snippet: The signal from the blots was detected using the G:BOX Chemi X system (Syngene, Cambridge, UK) and analyzed using ImageJ software. .. Western blotting was performed using the following antibodies: GAPDH (1:20,000, 60004-1-lg; Proteintech, Wuhan, China); β-actin (1:5,000, 60008-1-lg; Proteintech); SLC7A11/xCT (1:1,000, A2413; ABclonal, Wuhan, China); γ-H2AX (1:1,000, #9718; Cell Signaling Technology, Danvers, Massachusetts, USA); ATF4 (1:1,000, A18687; ABclonal); ATF3 (1:1,000, #18665; Cell Signaling Technology); NRF2 (1:1,000, #12721, Cell Signaling Technology); P53 (1:1,000, #2524; Cell Signaling Technology). .. GSH content in tumor cells was quantified using the corresponding kits (A005-1-2; Nanjing Jiancheng Bioengineering Institute, China) following the manufacturer’s protocol.

Article Title: ZNF423 depletion induces the integrated stress response and represents a potential vulnerability in NF1-associated MPNST
Article Snippet: Isolated proteins were fractioned using 4-20% Mini-PROTEAN TGX Stain-free gels (4568094, Bio-Rad) and electro-transferred to nitrocellulose membranes (1704158, Bio-Rad). .. Immunoblots were performed using primary antibodies against ZNF423 (ABN410, Sigma-Aldrich), GAPDH (sc-365062, Santa Cruz Biotechnology), SUZ12 (3737S, Cell Signaling Technology), ATF3 (18665S, Cell Signaling Technology), ATF4 (11815S, Cell Signaling Technology), PARP (9542S, Cell Signaling Technology), and γH2A.x (9718S, Cell Signaling Technology). .. Following incubation with primary antibodies at 4°C overnight, blots were washed 3x with TBST, before being incubated with appropriate horseradish peroxidase (HRP) – conjugated secondary antibodies, either anti-rabbit (7074V, Cell Signaling Technology) or anti-mouse (7076V, Cell Signaling Technology).

Incubation:

Article Title: Redox-dependent suppression of ATF3 impairs steroid sensitivity in asthma through MKP-1/p38 MAPK signaling.
Article Snippet: .. Sections were then incubated overnight at 4 ◦C with primary antibodies against ATF3 (Cell Signaling Technology, #18665, 1:150) and MKP-1 (Affinity Biosciences, AF5286, 1:200). .. After washing, sections were incubated with Opal Polymer HRPconjugated secondary antibody (Akoya Biosciences) for 10 min at room temperature, followed by Opal fluorescence working solution (Opal 520 for ATF3, Opal 690 for MKP-1) incubation for 10 min.

Article Title: Single-Cell and Machine Learning Analysis Reveal Novel Inflammatory Macrophage Subtypes and Biomarkers in Periodontitis
Article Snippet: .. Equal amounts of protein (30 μg/sample) were resolved by SDS-PAGE, transferred onto PVDF membranes and incubated overnight with primary antibodies specific for ATF3 (CST, #18665, 1:1500), CXCR4 (Abcam, ab181020, 1:1000), TXN (CST, #14907, 1:800), CBX3 (Abcam, ab217999, 1:1500), MBP (Abcam, ab11159, 1:800) and β-actin. ..

other:

Article Title: In vitro mimicking of humanized cardiogenesis under porcine condition
Article Snippet: Proteins were detected by an enhanced chemiluminescence kit (Vazyme E412-02).

Article Title: In vitro mimicking of humanized cardiogenesis under porcine condition.
Article Snippet: Proteins were detected by an enhanced chemiluminescence kit (Thermo).

Image Search Results

A375 parental and CTL-tolerant persister cell scRNA-seq analysis of (A) signature scores of melanoma cell states observed in patients (Mann-Whitney test), (B) GSEA of the antigen presentation melanoma cell state and (C) expression of selected antigen presentation cell state genes. (D) Proportion of genes in each melanoma cell state which are anastasis-associated genes and (E) A375 cell scRNA-seq expression of selected anastasis-associated genes. (F) ATF3 expression in A375 persisters ± 2 μM IDO1 inhibitor epacadostat or 100 μg/mL tryptophan supplementation added during coculture days 12-15. (G-H) IHC analysis of 4MOSC1 head and neck squamous cell carcinoma syngeneic tumors from mice treated with 10 mg/kg anti-PD-1. (G) Representative IHC images and (H) quantification (n = 4-5 mice, 5 regions were analyzed per tumor). Scale bars, 50 µm. (I-K) Analysis of surgically resected primary human cutaneous melanoma tissue treated with 10 μg/mL anti-PD-1, 10 ng/mL IFNγ, and 10 ng/mL TNF for 6 days in culture. (I) Representative IHC images and (J) quantification (n = 1, five regions were analyzed per tumor slice). Scale bars, 50 µm. (K) Treated primary melanoma cells with elevated caspase 3/7 activity were sorted, replated, and tested for viability 24 hours later by flow cytometry (n = 1). Mean ± SD are plotted and two-tailed unpaired t-tests were performed unless stated otherwise. ns P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. See also Figure S5 .

Journal: bioRxiv

Article Title: Antigenic cancer persister cells survive direct T cell attack

doi: 10.1101/2025.03.14.643359

Figure Lengend Snippet: A375 parental and CTL-tolerant persister cell scRNA-seq analysis of (A) signature scores of melanoma cell states observed in patients (Mann-Whitney test), (B) GSEA of the antigen presentation melanoma cell state and (C) expression of selected antigen presentation cell state genes. (D) Proportion of genes in each melanoma cell state which are anastasis-associated genes and (E) A375 cell scRNA-seq expression of selected anastasis-associated genes. (F) ATF3 expression in A375 persisters ± 2 μM IDO1 inhibitor epacadostat or 100 μg/mL tryptophan supplementation added during coculture days 12-15. (G-H) IHC analysis of 4MOSC1 head and neck squamous cell carcinoma syngeneic tumors from mice treated with 10 mg/kg anti-PD-1. (G) Representative IHC images and (H) quantification (n = 4-5 mice, 5 regions were analyzed per tumor). Scale bars, 50 µm. (I-K) Analysis of surgically resected primary human cutaneous melanoma tissue treated with 10 μg/mL anti-PD-1, 10 ng/mL IFNγ, and 10 ng/mL TNF for 6 days in culture. (I) Representative IHC images and (J) quantification (n = 1, five regions were analyzed per tumor slice). Scale bars, 50 µm. (K) Treated primary melanoma cells with elevated caspase 3/7 activity were sorted, replated, and tested for viability 24 hours later by flow cytometry (n = 1). Mean ± SD are plotted and two-tailed unpaired t-tests were performed unless stated otherwise. ns P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. See also Figure S5 .

Article Snippet: Primary antibodies against ATF3 (1:100, Cell Signaling Technology #18665), mouse IDO1 (1:200, LS-Bio #LS-B13059), human IDO1 (1:200, Cell Signaling Technology #86630), S100B (1:500, Abcam #ab52642), and pan-Cytokeratin (1:1000, Dako #Z0622) were used.

Techniques: MANN-WHITNEY, Expressing, Activity Assay, Flow Cytometry, Two Tailed Test

(A) Representative crystal violet staining and (B) microscopy depicting A375-A cells during one month of recombinant IFNγ ± TNF exposure (1 ng/ml each). Scalebars, 100 μm. (C) Western blot of A375 persister cells which survived 15 days of CTL low coculture or recombinant IFNγ ± TNF (1 ng/ml each) exposure. (D) A375 IFNγ-tolerant persister cells derived from 12 days of IFNγ exposure (1 ng/ml) were further treated from days 12-15 with IDO1 inhibitor (+IDO1i, 2 μM epacadostat) or tryptophan supplementation (+Trp, 100 μg/mL) with continued IFNγ exposure (t-tests versus IFNγ-only). (E) Western blot of ATF3 and ATF4 expression in A375 cytokine-persisters. (F) A375 parental cell viability after 6 days of cytokine treatment (t-tests versus untreated cells). (G) A375 cell viability after 15 days of recombinant IFNγ ± TNF exposure to form persister cells (t-tests versus 1 ng/ml IFNγ). (H) Western blot of IDO1 expression in A375 cytokine-persisters. (I) Quantification of A375-A EC formation after one month of IFNγ ± TNF exposure (1 ng/ml each). (J) Viability of A375 IFNγ + TNF-tolerant persister and EC cells regrown without cytokines and then rechallenged with 9 days of IFNγ + TNF exposure (1 ng/ml each) or (K) 6 days of CTL coculture (t-tests versus parental). The three cytokine-resistant ECs isolated from IFNγ ± TNF exposure of bulk A375 cells shown in J are the same ECs which are cross-resistant to CTL low exposure shown in K . N = 3, mean ± SD are plotted, and two-tailed unpaired t-tests were performed unless stated otherwise. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. See also Figure S9 .

Journal: bioRxiv

Article Title: Antigenic cancer persister cells survive direct T cell attack

doi: 10.1101/2025.03.14.643359

Figure Lengend Snippet: (A) Representative crystal violet staining and (B) microscopy depicting A375-A cells during one month of recombinant IFNγ ± TNF exposure (1 ng/ml each). Scalebars, 100 μm. (C) Western blot of A375 persister cells which survived 15 days of CTL low coculture or recombinant IFNγ ± TNF (1 ng/ml each) exposure. (D) A375 IFNγ-tolerant persister cells derived from 12 days of IFNγ exposure (1 ng/ml) were further treated from days 12-15 with IDO1 inhibitor (+IDO1i, 2 μM epacadostat) or tryptophan supplementation (+Trp, 100 μg/mL) with continued IFNγ exposure (t-tests versus IFNγ-only). (E) Western blot of ATF3 and ATF4 expression in A375 cytokine-persisters. (F) A375 parental cell viability after 6 days of cytokine treatment (t-tests versus untreated cells). (G) A375 cell viability after 15 days of recombinant IFNγ ± TNF exposure to form persister cells (t-tests versus 1 ng/ml IFNγ). (H) Western blot of IDO1 expression in A375 cytokine-persisters. (I) Quantification of A375-A EC formation after one month of IFNγ ± TNF exposure (1 ng/ml each). (J) Viability of A375 IFNγ + TNF-tolerant persister and EC cells regrown without cytokines and then rechallenged with 9 days of IFNγ + TNF exposure (1 ng/ml each) or (K) 6 days of CTL coculture (t-tests versus parental). The three cytokine-resistant ECs isolated from IFNγ ± TNF exposure of bulk A375 cells shown in J are the same ECs which are cross-resistant to CTL low exposure shown in K . N = 3, mean ± SD are plotted, and two-tailed unpaired t-tests were performed unless stated otherwise. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. See also Figure S9 .

Techniques: Staining, Microscopy, Recombinant, Western Blot, Derivative Assay, Expressing, Isolation, Two Tailed Test

Fig. 2 Grouped PCC and QCC populations exhibited distinct features and ATF3 is a hallmark of QCCs. A Scatterplot for hallmark pathways in the PCC population (Red dots indicate the top 3 signiﬁcantly changed pathways). B GSEA enrichment proﬁle of the top 3 altered hallmark pathways in the proliferated cell population, including Oxidative phosphorylation, MYC targets-V1, and Protein secretion. C Scatterplot for hallmark pathways in the QCC population (blue dots indicate the top 3 signiﬁcantly changed pathways). D GSEA enrichment proﬁle of the top 3 altered hallmark pathways in the quiescent cell population, including TNFα signaling via NF-kB, Hypoxia, and Cholesterol homeostasis. E Venn plot for overlapping genes involved in corresponding hallmark pathways in PCC and QCC populations. In the QCC population, ATF3, NFIL3 and PNRC1 are co-shared by the corresponding pathways: TNFα signaling via NF-kB, Hypoxia, and Cholesterol homeostasis. F Violin plots of gene expression of ATF3, NFIL3, and PNRC1 in Group 1 (PCCs) and Group 2 (QCCs) (****, p < 0.0001). G Expression levels of ATF3 and ATF4 in HCT116, HT-29, DLD-1 and HEK293T cells under both monolayer and MCTS conditions. H Immunochemistry staining for Ki67, p27 and ATF3 on HCT116 and HEK293T MCTS.

Journal: Cell death & disease

Article Title: Identification of ATF3 as a novel protective signature of quiescent colorectal tumor cells.

doi: 10.1038/s41419-023-06204-1

Figure Lengend Snippet: Fig. 2 Grouped PCC and QCC populations exhibited distinct features and ATF3 is a hallmark of QCCs. A Scatterplot for hallmark pathways in the PCC population (Red dots indicate the top 3 signiﬁcantly changed pathways). B GSEA enrichment proﬁle of the top 3 altered hallmark pathways in the proliferated cell population, including Oxidative phosphorylation, MYC targets-V1, and Protein secretion. C Scatterplot for hallmark pathways in the QCC population (blue dots indicate the top 3 signiﬁcantly changed pathways). D GSEA enrichment proﬁle of the top 3 altered hallmark pathways in the quiescent cell population, including TNFα signaling via NF-kB, Hypoxia, and Cholesterol homeostasis. E Venn plot for overlapping genes involved in corresponding hallmark pathways in PCC and QCC populations. In the QCC population, ATF3, NFIL3 and PNRC1 are co-shared by the corresponding pathways: TNFα signaling via NF-kB, Hypoxia, and Cholesterol homeostasis. F Violin plots of gene expression of ATF3, NFIL3, and PNRC1 in Group 1 (PCCs) and Group 2 (QCCs) (****, p < 0.0001). G Expression levels of ATF3 and ATF4 in HCT116, HT-29, DLD-1 and HEK293T cells under both monolayer and MCTS conditions. H Immunochemistry staining for Ki67, p27 and ATF3 on HCT116 and HEK293T MCTS.

Article Snippet: The ATF3 antibody (#18665) used for coimmunoprecipitation was obtained from Cell signaling (MA, USA).

Techniques: Phospho-proteomics, Gene Expression, Expressing, Staining

Fig. 3 QCCs could repopulate into PCCs. A Dynamic streams predicted by RNA velocity analysis with a dynamical model. Arrows indicate the direction of the change in cell status based on the mRNA maturation in QCC and PCC groups. B Pseudotime predicted by the same workﬂow in (A). C A heatmap representation of the pseudotime inference shown in (A) and (B) in QCC and PCC groups. D Gene expression and phase portrait of cell-cycle activity markers MKI67 and CDK4. E Gene expression and splicing rate of cell-cycle inactive markers CDKN1A and CDKN1C. F Gene expression and splicing rate of QCCs-dependent markers ATF3.

Journal: Cell death & disease

Article Title: Identification of ATF3 as a novel protective signature of quiescent colorectal tumor cells.

doi: 10.1038/s41419-023-06204-1

Figure Lengend Snippet: Fig. 3 QCCs could repopulate into PCCs. A Dynamic streams predicted by RNA velocity analysis with a dynamical model. Arrows indicate the direction of the change in cell status based on the mRNA maturation in QCC and PCC groups. B Pseudotime predicted by the same workﬂow in (A). C A heatmap representation of the pseudotime inference shown in (A) and (B) in QCC and PCC groups. D Gene expression and phase portrait of cell-cycle activity markers MKI67 and CDK4. E Gene expression and splicing rate of cell-cycle inactive markers CDKN1A and CDKN1C. F Gene expression and splicing rate of QCCs-dependent markers ATF3.

Article Snippet: The ATF3 antibody (#18665) used for coimmunoprecipitation was obtained from Cell signaling (MA, USA).

Techniques: Gene Expression, Activity Assay

Fig. 4 Cells lacking ATF3 show upregulated mitochondrial function and are more sensitive to anticancer agents. A Expression levels of ATF3 in ATF3parental and ATF3KO cells under monolayer and MCTS conditions. Actin is the loading control. Three independent experiments were performed. B Images of ATF3parental and ATF3KO MCTS taken by Incucyte, Scale bar: 300 µm. C GSEA enrichment proﬁle of the top-altered hallmark pathways in HCT116 MCTS lacking ATF3 compared with HCT116 ATF3parental MCTS. D Reactome analysis showing upregulation of the respiratory electron transport pathway and mitochondrial translation elongation in ATF3-deﬁcient HCT116 MCTS. Table lists genes involved in complex I biogenesis and mitochondrial translation elongation. E Flow chart of hit compound identiﬁcation using mechanistic screening set (Library description: Mechanistic Set VI, https://dtp.cancer.gov). F NSC numbers and chemical structures of the hit compounds. G Dose response to hit compounds in HCT116 ATF3parental and ATF3KO cells under monolayer conditions. The half-maximal inhibitory concentration (IC50) was calculated using nonlinear regression, [Inhibitor] vs. normalized response analysis. H Dose response to hit compounds in HT-29 ATF3scramble and ATF3KD cells under monolayer conditions. The half-maximal inhibitory concentration (IC50) was calculated using nonlinear regression, [Inhibitor] vs. normalized response analysis. I Expression levels of ATF3 in HCT116, DLD-1 and HT-29 cells treated with DMSO or 100 nM sangivamycin for 24 h under monolayer conditions. Actin is the loading control. J Expression levels of ATF3 in ATF3parental and ATF3KO cells treated with DMSO or 100 nM sangivamycin for 24 h under MCTS conditions. Actin is the loading control. K Dose response to sangivamycin. HCT116 ATF3parental and ATF3KO cells were treated with sangivamycin in monolayer condition for 72 h. The data is expressed as the mean ± SD (n = 3, unpaired t-test, two-stage step-up (Benjamini, Krieger, and Yekutieli); * p < 0.05, ** p < 0.01, *** p < 0.001). The half- maximal inhibitory concentration (IC50) was calculated using nonlinear regression, [Inhibitor] vs. normalized response analysis. L MCTS treated with indicated concentrations of sangivamycin was monitored in Incucyte for 3 days. Images for each spheroid were taken every day. The volume of spheroids was calculated by equation Volume = (4/3)*3.14*R3 (R = radius). The data is expressed as the mean ± SD (n = 3, using multiple-unpaired t-test, * p < 0.05).

Journal: Cell death & disease

Article Title: Identification of ATF3 as a novel protective signature of quiescent colorectal tumor cells.

doi: 10.1038/s41419-023-06204-1

Figure Lengend Snippet: Fig. 4 Cells lacking ATF3 show upregulated mitochondrial function and are more sensitive to anticancer agents. A Expression levels of ATF3 in ATF3parental and ATF3KO cells under monolayer and MCTS conditions. Actin is the loading control. Three independent experiments were performed. B Images of ATF3parental and ATF3KO MCTS taken by Incucyte, Scale bar: 300 µm. C GSEA enrichment proﬁle of the top-altered hallmark pathways in HCT116 MCTS lacking ATF3 compared with HCT116 ATF3parental MCTS. D Reactome analysis showing upregulation of the respiratory electron transport pathway and mitochondrial translation elongation in ATF3-deﬁcient HCT116 MCTS. Table lists genes involved in complex I biogenesis and mitochondrial translation elongation. E Flow chart of hit compound identiﬁcation using mechanistic screening set (Library description: Mechanistic Set VI, https://dtp.cancer.gov). F NSC numbers and chemical structures of the hit compounds. G Dose response to hit compounds in HCT116 ATF3parental and ATF3KO cells under monolayer conditions. The half-maximal inhibitory concentration (IC50) was calculated using nonlinear regression, [Inhibitor] vs. normalized response analysis. H Dose response to hit compounds in HT-29 ATF3scramble and ATF3KD cells under monolayer conditions. The half-maximal inhibitory concentration (IC50) was calculated using nonlinear regression, [Inhibitor] vs. normalized response analysis. I Expression levels of ATF3 in HCT116, DLD-1 and HT-29 cells treated with DMSO or 100 nM sangivamycin for 24 h under monolayer conditions. Actin is the loading control. J Expression levels of ATF3 in ATF3parental and ATF3KO cells treated with DMSO or 100 nM sangivamycin for 24 h under MCTS conditions. Actin is the loading control. K Dose response to sangivamycin. HCT116 ATF3parental and ATF3KO cells were treated with sangivamycin in monolayer condition for 72 h. The data is expressed as the mean ± SD (n = 3, unpaired t-test, two-stage step-up (Benjamini, Krieger, and Yekutieli); * p < 0.05, ** p < 0.01, *** p < 0.001). The half- maximal inhibitory concentration (IC50) was calculated using nonlinear regression, [Inhibitor] vs. normalized response analysis. L MCTS treated with indicated concentrations of sangivamycin was monitored in Incucyte for 3 days. Images for each spheroid were taken every day. The volume of spheroids was calculated by equation Volume = (4/3)*3.14*R3 (R = radius). The data is expressed as the mean ± SD (n = 3, using multiple-unpaired t-test, * p < 0.05).

Article Snippet: The ATF3 antibody (#18665) used for coimmunoprecipitation was obtained from Cell signaling (MA, USA).

Techniques: Expressing, Control, Concentration Assay

Fig. 5 ATF3 could interact with TRIB3 and DDIT4 at the chromatin level. A Schematic illustration of the overall experimental designs. ATAC- Seq stands for Assay for Transposase-Accessible Chromatin with high-throughput sequencing. The ATAC-Seq method relies on next- generation sequencing (NGS) library construction using the hyperactive transposase Tn5. NGS adapters are loaded onto the transposase, which allows simultaneous fragmentation of chromatin and integration of those adapters into open chromatin regions. The libraries generated from cells treated with DMSO or 100 nM sangivamycin for 24 h were sequenced by NGS, and the regions of the genome with open or accessible chromatin are analyzed using bioinformatics. B Enrichment score of ATAC-Seq samples at transcription start sites (TSS) from treated and untreated cells. C Fraction of reads in peaks (FRiP) in duplicate samples. The dashed line shows the minimum FRiP score required for further analysis of that sample. D Volcano plot for enrichment transcription factors in the sangivamycin (100 nM, 24 h) treated group. E Heatmap of unique accessible chromatin regions of two groups treated with or without sangivamycin (100 nM, 24 h). Representative genes annotated to the chromatin regions are indicated. F Genome browser tracks of ATAC-Seq data for each sample of ATF3, ATF4, TRIB3 and DDIT4. Tracks show signiﬁcant peak signal intensity (y-axis) for open chromatin regions of indicated genes (TRIB3 and DDIT4) (x-axis) in treated groups (pink, bottom) compared to the control groups (blue, top). Individual gene maps are shown in dark blue. G Scatter plot for enrichment transcription factors in the Sangivamycin group. ATF3 was one of the top-altered TFs.

Journal: Cell death & disease

Article Title: Identification of ATF3 as a novel protective signature of quiescent colorectal tumor cells.

doi: 10.1038/s41419-023-06204-1

Figure Lengend Snippet: Fig. 5 ATF3 could interact with TRIB3 and DDIT4 at the chromatin level. A Schematic illustration of the overall experimental designs. ATAC- Seq stands for Assay for Transposase-Accessible Chromatin with high-throughput sequencing. The ATAC-Seq method relies on next- generation sequencing (NGS) library construction using the hyperactive transposase Tn5. NGS adapters are loaded onto the transposase, which allows simultaneous fragmentation of chromatin and integration of those adapters into open chromatin regions. The libraries generated from cells treated with DMSO or 100 nM sangivamycin for 24 h were sequenced by NGS, and the regions of the genome with open or accessible chromatin are analyzed using bioinformatics. B Enrichment score of ATAC-Seq samples at transcription start sites (TSS) from treated and untreated cells. C Fraction of reads in peaks (FRiP) in duplicate samples. The dashed line shows the minimum FRiP score required for further analysis of that sample. D Volcano plot for enrichment transcription factors in the sangivamycin (100 nM, 24 h) treated group. E Heatmap of unique accessible chromatin regions of two groups treated with or without sangivamycin (100 nM, 24 h). Representative genes annotated to the chromatin regions are indicated. F Genome browser tracks of ATAC-Seq data for each sample of ATF3, ATF4, TRIB3 and DDIT4. Tracks show signiﬁcant peak signal intensity (y-axis) for open chromatin regions of indicated genes (TRIB3 and DDIT4) (x-axis) in treated groups (pink, bottom) compared to the control groups (blue, top). Individual gene maps are shown in dark blue. G Scatter plot for enrichment transcription factors in the Sangivamycin group. ATF3 was one of the top-altered TFs.

Article Snippet: The ATF3 antibody (#18665) used for coimmunoprecipitation was obtained from Cell signaling (MA, USA).

Techniques: Next-Generation Sequencing, Generated, Control

Fig. 6 Lowering ATF3 improves the effect of 5-FU on CRC MCTS. A Plot for protein interactions among genes ATF3, ATF4, DDIT4, TRIB3 and FGD6 by the STRING interaction network. B Expression levels of ATF3, ATF4, DDIT4 and TRIB3 in HCT116, DLD-1 and HT-29 cells treated with DMSO or 100 nM sangivamycin for 24 h under monolayer conditions. Actin is the loading control. C Expression levels of ATF3, DDIT4 and TRIB3 were assessed in COAD tissues and normal adjacent tissues using RNA-Seq data from TCGA (* p < 0.05). Cohort size: (num)normal adjacent tissues = 349; (num) COAD tissues = 275. D Western blot analysis of the co-IP complex from cells treated with DMSO or 100 nM sangivamycin for 24 h with ATF3 antibody immobilized on Dynabeads®. Tubulin is the loading control for the input conditions. E Expression levels of ATF3, ATF4, DDIT4 and TRIB3 in HCT116 ATF3parental and ATF3KO cells treated with DMSO or 100 nM sangivamycin for 24 h under monolayer conditions. Actin is the loading control. F Illustration of the interaction between ATF3, ATF4, DDIT4 and TRIB3 based on our results and ﬁndings from other groups [79–81]. G ATF3 expression levels in HCT116 and HT-29 cells treated with the indicated concentrations of rapamycin for 72 h under monolayer conditions. Actin is the loading control. H ATF3 expression levels in HCT116 MCTS cells treated with rapamycin (10 µM) or Torin-1 (0.5 µM) for 72 h. Actin is the loading control. I Dose response of HCT116 and HT29 MCTS to 5-FU. The data is expressed as the mean ± SD (n = 3). The half-maximal inhibitory concentration (IC50) was calculated using nonlinear regression, [Inhibitor] vs. normalized response analysis. J Combination of rapamycin (10 µM) or Troin1(0.5 µM) with 5-FU under HCT116 MCTS conditions for 72 h. The data is expressed as the mean ± SD (n = 3, unpaired t-test, two-stage step-up (Benjamini, Krieger, and Yekutieli); * p < 0.05, *** p < 0.001). K Combination of rapamycin (10 µM) or Troin1(0.5 µM) with 5-FU under HT29 MCTS conditions for 72 h. CDI = AB/(A × B) where: AB = cell viability value for the combination of rapamycin/torin-1 and 5-FU. A and B = cell viability value for the single treatment rapamycin/torin-1 or 5-FU. A CDI value <1, =1 or >1 indicates that the drugs are synergistic, additive, or antagonistic, respectively. A CDI value less than 0.7 indicates a signiﬁcant synergetic effect [31].

Journal: Cell death & disease

Article Title: Identification of ATF3 as a novel protective signature of quiescent colorectal tumor cells.

doi: 10.1038/s41419-023-06204-1

Figure Lengend Snippet: Fig. 6 Lowering ATF3 improves the effect of 5-FU on CRC MCTS. A Plot for protein interactions among genes ATF3, ATF4, DDIT4, TRIB3 and FGD6 by the STRING interaction network. B Expression levels of ATF3, ATF4, DDIT4 and TRIB3 in HCT116, DLD-1 and HT-29 cells treated with DMSO or 100 nM sangivamycin for 24 h under monolayer conditions. Actin is the loading control. C Expression levels of ATF3, DDIT4 and TRIB3 were assessed in COAD tissues and normal adjacent tissues using RNA-Seq data from TCGA (* p < 0.05). Cohort size: (num)normal adjacent tissues = 349; (num) COAD tissues = 275. D Western blot analysis of the co-IP complex from cells treated with DMSO or 100 nM sangivamycin for 24 h with ATF3 antibody immobilized on Dynabeads®. Tubulin is the loading control for the input conditions. E Expression levels of ATF3, ATF4, DDIT4 and TRIB3 in HCT116 ATF3parental and ATF3KO cells treated with DMSO or 100 nM sangivamycin for 24 h under monolayer conditions. Actin is the loading control. F Illustration of the interaction between ATF3, ATF4, DDIT4 and TRIB3 based on our results and ﬁndings from other groups [79–81]. G ATF3 expression levels in HCT116 and HT-29 cells treated with the indicated concentrations of rapamycin for 72 h under monolayer conditions. Actin is the loading control. H ATF3 expression levels in HCT116 MCTS cells treated with rapamycin (10 µM) or Torin-1 (0.5 µM) for 72 h. Actin is the loading control. I Dose response of HCT116 and HT29 MCTS to 5-FU. The data is expressed as the mean ± SD (n = 3). The half-maximal inhibitory concentration (IC50) was calculated using nonlinear regression, [Inhibitor] vs. normalized response analysis. J Combination of rapamycin (10 µM) or Troin1(0.5 µM) with 5-FU under HCT116 MCTS conditions for 72 h. The data is expressed as the mean ± SD (n = 3, unpaired t-test, two-stage step-up (Benjamini, Krieger, and Yekutieli); * p < 0.05, *** p < 0.001). K Combination of rapamycin (10 µM) or Troin1(0.5 µM) with 5-FU under HT29 MCTS conditions for 72 h. CDI = AB/(A × B) where: AB = cell viability value for the combination of rapamycin/torin-1 and 5-FU. A and B = cell viability value for the single treatment rapamycin/torin-1 or 5-FU. A CDI value <1, =1 or >1 indicates that the drugs are synergistic, additive, or antagonistic, respectively. A CDI value less than 0.7 indicates a signiﬁcant synergetic effect [31].

Article Snippet: The ATF3 antibody (#18665) used for coimmunoprecipitation was obtained from Cell signaling (MA, USA).

Techniques: Expressing, Control, RNA Sequencing, Western Blot, Co-Immunoprecipitation Assay, Concentration Assay

Activation of the ATF4-ATF3 pathway is crucial for CHMP2A loss-induced cell death. (A) Western blot analysis of the indicated cells that were treated with DMSO or 30 nM THG for 24 h. (B, C) Quantification of active CASP3- or CASP7-positive (B) and YOYO-3 iodide-positive dead (C) cells after transfection with the indicated siRNAs. (D) Western blot analysis of the indicated i-shRNA-expressing U-2 OS cells were pretreated with 1 µg/ml Dox for 48 h followed by the treatment with 1 µM GSK157 for 24 h in the presence of 1 µg/ml Dox. (E) Quantification of YOYO-3 iodide-positive dead U-2 OS cells expressing the indicated i-shRNA after pretreatment with 1 µg/ml Dox for 48 h followed by treatment with 1 µM GSK157 or DMSO in presence of 1 µg/ml Dox. (F) Quantification of YOYO-3 iodide-positive dead U-2 OS cells expressing the indicated i-shRNA after pretreatment with 1 µg/ml Dox for 24 h followed by transfection of si RELA or non-targeting siRNA in presence of 1 µg/ml Dox. Western blot analysis data demonstrating successful target gene depletion after 48 h post transfection was shown on the top. In B, C, E and F, IncuCyte images at the experimental endpoints are shown on the right. Scale bars: 100 μm. All values in the graphs are mean ± SD ( n = 3 from three independent experiments).

Journal: Autophagy

Article Title: ER stress elicits non-canonical CASP8 (caspase 8) activation on autophagosomal membranes to induce apoptosis

doi: 10.1080/15548627.2023.2258701

Figure Lengend Snippet: Activation of the ATF4-ATF3 pathway is crucial for CHMP2A loss-induced cell death. (A) Western blot analysis of the indicated cells that were treated with DMSO or 30 nM THG for 24 h. (B, C) Quantification of active CASP3- or CASP7-positive (B) and YOYO-3 iodide-positive dead (C) cells after transfection with the indicated siRNAs. (D) Western blot analysis of the indicated i-shRNA-expressing U-2 OS cells were pretreated with 1 µg/ml Dox for 48 h followed by the treatment with 1 µM GSK157 for 24 h in the presence of 1 µg/ml Dox. (E) Quantification of YOYO-3 iodide-positive dead U-2 OS cells expressing the indicated i-shRNA after pretreatment with 1 µg/ml Dox for 48 h followed by treatment with 1 µM GSK157 or DMSO in presence of 1 µg/ml Dox. (F) Quantification of YOYO-3 iodide-positive dead U-2 OS cells expressing the indicated i-shRNA after pretreatment with 1 µg/ml Dox for 24 h followed by transfection of si RELA or non-targeting siRNA in presence of 1 µg/ml Dox. Western blot analysis data demonstrating successful target gene depletion after 48 h post transfection was shown on the top. In B, C, E and F, IncuCyte images at the experimental endpoints are shown on the right. Scale bars: 100 μm. All values in the graphs are mean ± SD ( n = 3 from three independent experiments).

Article Snippet: The following antibodies were used for immunofluorescence (IF) and immunoblotting (IB): mouse antibodies against ACTB/β-ACTIN (Sigma-Aldrich, A5441; IB, 1:10,000), DDIT3 (Cell Signaling Technology, 2895; IB, 1:1,000), NFKBIA (Cell Signaling Technology, 4814; IB, 1:1,000), VPS37A (Santa Cruz Biotechnology, sc-376978; IB, 1:100); rabbit antibodies against ATF3 (Cell Signaling Technology, 18665; IB, 1:1,000), ATF4 (Cell Signaling Technology, 11815; IB, 1:1,000), ATG5 (Cell Signaling Technology, 12994; IB, 1:1,000), ATG7 (Cell Signaling Technology, 8558; IB, 1:1,000), BCL2L1 (Cell Signaling Technology, 2762; IB, 1:1,000), HSPA5/BiP (Cell Signaling Technology, 3177; IB, 1:1,000), CHMP2A (Proteintech, 10477-1-AP; IB, 1:1,000), cleaved CASP3 (Cell Signaling Technology, 9661; IB, 1:1,000), cleaved CASP8 (Cell Signaling Technology, 9496; IB, 1:1,000), CASP9 (Cell Signaling Technology, 9502; IB, 1:1,000), TNFRSF10B/DR5 (Cell Signaling Technology, 8074; IB, 1:1,000), GAPDH (Proteintech, 60004-1-lg; IB, 1:50,000), HRK (Novus, NBP1-76414; IB, 1:400), LMNB1 (Proteintech, 12987-1-AP; IB, 1:5,000), MAP1LC3B (Novus, NB100-2220; IB, 1:5,000) (Cell Signaling Technology, 3868; IF, 1:200), PARP (Cell Signaling Technology, 9542; IB, 1:1,000), PMAIP1 (Cell Signaling Technology, 14766; IB, 1:1,000), CASP8 (Abcam, ab108333; IB, 1:1,000), RELA (Cell Signaling Technology, 8242; IB, 1:1,000), TRIB3 (Proteintech, 13300-1-AP; IB, 1:1,000); guinea pig antibody against SQSTM1/p62 (American Research Products, 03-GP62-C; IB, 1:4,000; IF, 1:400).

Techniques: Activation Assay, Western Blot, Transfection, shRNA, Expressing

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