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primary antibodies against atf3  (Thermo Fisher)


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    Structured Review

    Thermo Fisher primary antibodies against atf3
    Primary Antibodies Against Atf3, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/primary antibodies against atf3/product/Thermo Fisher
    Average 90 stars, based on 1 article reviews
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    Cell Signaling Technology Inc primary antibodies against atf3
    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) <t>ATF3</t> 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 .
    Primary Antibodies Against Atf3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    RT-QPCR showed the expression of <t>SCD1</t> and its regulatory genes ATF3 and CCL4 expression in tumor tissues of mice (n = 3) (A–C). The expression of SCD1, ATF3, CCL4 and infiltration of CD8+T cells were detected in tumor tissues of mice in different treatment groups by IHC (D–H). The protein levels of SCD1, ATF3 and CCL4 in mouse tumor tissues were detected by western blotting (I–L).
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    A , B RNA-seq results showed that <t>ATF3</t> in the endometrium of RIF patients was downregulated (RIF) compared with that in fertile controls (FER). C ATF3 expression in RIF and FER endometrium was measured by quantitative PCR (qPCR). The expression of ATF3 was normalized to 18S rRNA expression, and the results are presented relative to FER stage ( n = 14 for FER group and n = 16 for RIF group). **** P < 0.0001 compared with the FER group. D , E Immunoblotting for ATF3 in the endometrium of RIF patients and the FER group. Quantitative densitometry analysis indicated that ATF3 levels were downregulated in RIF patients (n = 51). **** P < 0.0001 compared with the FER group. F , G Representative immunohistochemical images depicting the expression of ATF3 in the endometrium from fertile women and RIF patients. The negative control (NC) was nonspecific rabbit serum. The H -score of ATF3 expression in the endometrial stromal cells was calculated with IHC-Profiler and ImageJ (scale bar = 50 µm, n = 6, *** P < 0.001). H qPCR was performed to detect the expression of PRL in the endometrium of RIF patients, normalized to 18S rRNA expression ( n = 6).
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    A , B RNA-seq results showed that <t>ATF3</t> in the endometrium of RIF patients was downregulated (RIF) compared with that in fertile controls (FER). C ATF3 expression in RIF and FER endometrium was measured by quantitative PCR (qPCR). The expression of ATF3 was normalized to 18S rRNA expression, and the results are presented relative to FER stage ( n = 14 for FER group and n = 16 for RIF group). **** P < 0.0001 compared with the FER group. D , E Immunoblotting for ATF3 in the endometrium of RIF patients and the FER group. Quantitative densitometry analysis indicated that ATF3 levels were downregulated in RIF patients (n = 51). **** P < 0.0001 compared with the FER group. F , G Representative immunohistochemical images depicting the expression of ATF3 in the endometrium from fertile women and RIF patients. The negative control (NC) was nonspecific rabbit serum. The H -score of ATF3 expression in the endometrial stromal cells was calculated with IHC-Profiler and ImageJ (scale bar = 50 µm, n = 6, *** P < 0.001). H qPCR was performed to detect the expression of PRL in the endometrium of RIF patients, normalized to 18S rRNA expression ( n = 6).
    Primary Specific Antibodies Against Nf κb (P65), Iκbα, P Erk, Erk, P Jnk, Jnk, P P38, P38, Atf3, βactin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    (A) Computational predicting binding sites of <t>ATF3</t> and other transcription factors were shown in the up-stream of these genes. (B) Experimental results of ATF3 ChIP-seq and peaks for ATF3 binding sites were show in Integrative Genomics Viewer (IGV).
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    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 .

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

    RT-QPCR showed the expression of SCD1 and its regulatory genes ATF3 and CCL4 expression in tumor tissues of mice (n = 3) (A–C). The expression of SCD1, ATF3, CCL4 and infiltration of CD8+T cells were detected in tumor tissues of mice in different treatment groups by IHC (D–H). The protein levels of SCD1, ATF3 and CCL4 in mouse tumor tissues were detected by western blotting (I–L).

    Journal: Heliyon

    Article Title: CDK4/6i enhances the antitumor effect of PD1 antibody by promoting TLS formation in ovarian cancer

    doi: 10.1016/j.heliyon.2023.e19760

    Figure Lengend Snippet: RT-QPCR showed the expression of SCD1 and its regulatory genes ATF3 and CCL4 expression in tumor tissues of mice (n = 3) (A–C). The expression of SCD1, ATF3, CCL4 and infiltration of CD8+T cells were detected in tumor tissues of mice in different treatment groups by IHC (D–H). The protein levels of SCD1, ATF3 and CCL4 in mouse tumor tissues were detected by western blotting (I–L).

    Article Snippet: After blocking the membranes with 5% bovine serum albumin, the primary antibodies against SCD1 (1:2000, Affinity, DF13253), ATF3 (1:2000, Signalway Antibody, #53615), CCL4 (1:2000, Affinity, DF6545), and β-actin (1:10000, Abmart, T40104) were incubated at 4 °C overnight.

    Techniques: Quantitative RT-PCR, Expressing, Western Blot

    IHC results displayed correlation analysis between SCD1 expression and TLS score in ovarian cancer patients (A–B).

    Journal: Heliyon

    Article Title: CDK4/6i enhances the antitumor effect of PD1 antibody by promoting TLS formation in ovarian cancer

    doi: 10.1016/j.heliyon.2023.e19760

    Figure Lengend Snippet: IHC results displayed correlation analysis between SCD1 expression and TLS score in ovarian cancer patients (A–B).

    Article Snippet: After blocking the membranes with 5% bovine serum albumin, the primary antibodies against SCD1 (1:2000, Affinity, DF13253), ATF3 (1:2000, Signalway Antibody, #53615), CCL4 (1:2000, Affinity, DF6545), and β-actin (1:10000, Abmart, T40104) were incubated at 4 °C overnight.

    Techniques: Expressing

    A , B RNA-seq results showed that ATF3 in the endometrium of RIF patients was downregulated (RIF) compared with that in fertile controls (FER). C ATF3 expression in RIF and FER endometrium was measured by quantitative PCR (qPCR). The expression of ATF3 was normalized to 18S rRNA expression, and the results are presented relative to FER stage ( n = 14 for FER group and n = 16 for RIF group). **** P < 0.0001 compared with the FER group. D , E Immunoblotting for ATF3 in the endometrium of RIF patients and the FER group. Quantitative densitometry analysis indicated that ATF3 levels were downregulated in RIF patients (n = 51). **** P < 0.0001 compared with the FER group. F , G Representative immunohistochemical images depicting the expression of ATF3 in the endometrium from fertile women and RIF patients. The negative control (NC) was nonspecific rabbit serum. The H -score of ATF3 expression in the endometrial stromal cells was calculated with IHC-Profiler and ImageJ (scale bar = 50 µm, n = 6, *** P < 0.001). H qPCR was performed to detect the expression of PRL in the endometrium of RIF patients, normalized to 18S rRNA expression ( n = 6).

    Journal: Cell Death & Disease

    Article Title: ATF3 deficiency impairs the proliferative–secretory phase transition and decidualization in RIF patients

    doi: 10.1038/s41419-021-03679-8

    Figure Lengend Snippet: A , B RNA-seq results showed that ATF3 in the endometrium of RIF patients was downregulated (RIF) compared with that in fertile controls (FER). C ATF3 expression in RIF and FER endometrium was measured by quantitative PCR (qPCR). The expression of ATF3 was normalized to 18S rRNA expression, and the results are presented relative to FER stage ( n = 14 for FER group and n = 16 for RIF group). **** P < 0.0001 compared with the FER group. D , E Immunoblotting for ATF3 in the endometrium of RIF patients and the FER group. Quantitative densitometry analysis indicated that ATF3 levels were downregulated in RIF patients (n = 51). **** P < 0.0001 compared with the FER group. F , G Representative immunohistochemical images depicting the expression of ATF3 in the endometrium from fertile women and RIF patients. The negative control (NC) was nonspecific rabbit serum. The H -score of ATF3 expression in the endometrial stromal cells was calculated with IHC-Profiler and ImageJ (scale bar = 50 µm, n = 6, *** P < 0.001). H qPCR was performed to detect the expression of PRL in the endometrium of RIF patients, normalized to 18S rRNA expression ( n = 6).

    Article Snippet: Immunoblotting was performed by incubating the membranes with primary antibodies against ATF3 (1:1000; HPA001562, Sigma), FOXO1 (1:2000; Cell Signaling Technology, Danvers, MA, United States), His-Tag (1:2000; M30111, Abmart, Shanghai, China), and GAPDH (1:10,000; AP0063, Bioworld Technology, MN, USA).

    Techniques: RNA Sequencing Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunohistochemical staining, Negative Control

    A , B Immunohistochemistry analysis with an anti-ATF3 antibody. Proliferative and mid-secretory phase endometrial tissue samples from normal fertile women. The negative control (NC) was nonspecific rabbit serum. The H -score of ATF3 expression in the endometrial stromal cells was calculated with IHC-Profiler and ImageJ (Scale bar = 50 µm, n = 3, * P < 0.05). C Expression of ATF3 in proliferative and early-, mid-, and late-luteal phase endometrium. Each bar represents an individual biopsy. The data were retrieved from microarray data deposited in the Gene Expression Omnibus (GDS2052). D , E ATF3 expression pattern analyzed with a recent single-cell RNA-seq analysis in stromal cells ( D ) and all cells ( E ). The data were retrieved from microarray data deposited in the Gene Expression Omnibus (GSE111976). F The expression pattern of ATF3 in hESCs treated with 0.5 mM 8-Br-cAMP and 1 μM MPA (M + A) for different periods of time (0, 0.5, 1, 2, 4, 8, or 16 h) was evaluated by qPCR. * P < 0.05, ** P < 0.01. G The expression pattern of ATF3 in hESCs treated with 0.5 mM 8-Br-cAMP and 1 μM MPA (M + A) for different periods of time (0, 1, 3, 6, 12, 24, 48, or 72 h) was evaluated by western blot. H GSEA results showed that the ATF3-regulated gene term was enriched during in vitro decidualization. I , J hESCs were transfected with siATF3 or siCtl for 48 h and then treated with a decidualization stimulus. The expression and secretion of PRL were measured by qPCR and ELISA, respectively. * P < 0.05, ** P < 0.01 compared with the CTL/M + A group (8-Br-cAMP+MPA). K Immunofluorescence was performed to analyze the morphological transformation of hESCs.

    Journal: Cell Death & Disease

    Article Title: ATF3 deficiency impairs the proliferative–secretory phase transition and decidualization in RIF patients

    doi: 10.1038/s41419-021-03679-8

    Figure Lengend Snippet: A , B Immunohistochemistry analysis with an anti-ATF3 antibody. Proliferative and mid-secretory phase endometrial tissue samples from normal fertile women. The negative control (NC) was nonspecific rabbit serum. The H -score of ATF3 expression in the endometrial stromal cells was calculated with IHC-Profiler and ImageJ (Scale bar = 50 µm, n = 3, * P < 0.05). C Expression of ATF3 in proliferative and early-, mid-, and late-luteal phase endometrium. Each bar represents an individual biopsy. The data were retrieved from microarray data deposited in the Gene Expression Omnibus (GDS2052). D , E ATF3 expression pattern analyzed with a recent single-cell RNA-seq analysis in stromal cells ( D ) and all cells ( E ). The data were retrieved from microarray data deposited in the Gene Expression Omnibus (GSE111976). F The expression pattern of ATF3 in hESCs treated with 0.5 mM 8-Br-cAMP and 1 μM MPA (M + A) for different periods of time (0, 0.5, 1, 2, 4, 8, or 16 h) was evaluated by qPCR. * P < 0.05, ** P < 0.01. G The expression pattern of ATF3 in hESCs treated with 0.5 mM 8-Br-cAMP and 1 μM MPA (M + A) for different periods of time (0, 1, 3, 6, 12, 24, 48, or 72 h) was evaluated by western blot. H GSEA results showed that the ATF3-regulated gene term was enriched during in vitro decidualization. I , J hESCs were transfected with siATF3 or siCtl for 48 h and then treated with a decidualization stimulus. The expression and secretion of PRL were measured by qPCR and ELISA, respectively. * P < 0.05, ** P < 0.01 compared with the CTL/M + A group (8-Br-cAMP+MPA). K Immunofluorescence was performed to analyze the morphological transformation of hESCs.

    Article Snippet: Immunoblotting was performed by incubating the membranes with primary antibodies against ATF3 (1:1000; HPA001562, Sigma), FOXO1 (1:2000; Cell Signaling Technology, Danvers, MA, United States), His-Tag (1:2000; M30111, Abmart, Shanghai, China), and GAPDH (1:10,000; AP0063, Bioworld Technology, MN, USA).

    Techniques: Immunohistochemistry, Negative Control, Expressing, Microarray, RNA Sequencing Assay, Western Blot, In Vitro, Transfection, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Transformation Assay

    A Heat map showing hierarchical clustering of gene expression in decidualization and knockdown of the ATF3 group. B , C KEGG pathway enrichment analysis and GO analysis of the DEGs. D The DEGs related to the altered KEGG pathways. E GSEA results showed that the FoxO-targeted gene term was downregulated when ATF3 was silenced. F The relative expression of FoxO1 target genes, such as PRL, IGFBP-1, DCN, and LEFTY2.

    Journal: Cell Death & Disease

    Article Title: ATF3 deficiency impairs the proliferative–secretory phase transition and decidualization in RIF patients

    doi: 10.1038/s41419-021-03679-8

    Figure Lengend Snippet: A Heat map showing hierarchical clustering of gene expression in decidualization and knockdown of the ATF3 group. B , C KEGG pathway enrichment analysis and GO analysis of the DEGs. D The DEGs related to the altered KEGG pathways. E GSEA results showed that the FoxO-targeted gene term was downregulated when ATF3 was silenced. F The relative expression of FoxO1 target genes, such as PRL, IGFBP-1, DCN, and LEFTY2.

    Article Snippet: Immunoblotting was performed by incubating the membranes with primary antibodies against ATF3 (1:1000; HPA001562, Sigma), FOXO1 (1:2000; Cell Signaling Technology, Danvers, MA, United States), His-Tag (1:2000; M30111, Abmart, Shanghai, China), and GAPDH (1:10,000; AP0063, Bioworld Technology, MN, USA).

    Techniques: Expressing

    A hESCs were treated with Ad-LacZ or Ad-ATF3-His for 48 h and transfected with IRS-luc and Rinella for dual-luciferase assays. **** P < 0.0001. B – D hESCs were transfected with Ad-ATF3-his (0 or 40 MOI) or Ad-LacZ. si-FOXO1 was used to knockdown the expression of FOXO1, and prolactin secretion was measured by an enzyme-linked fluorescent assay (ELFA). * P < 0.05. Immunofluorescence was performed to analyze the morphological transformation of hESCs. Scale bar = 50 μm. E hESCs were transfected with Ad-ATF3-his (0, 20, or 40 MOI) or Ad-LacZ. FOXO1 and His protein expression levels were measured by western blot.

    Journal: Cell Death & Disease

    Article Title: ATF3 deficiency impairs the proliferative–secretory phase transition and decidualization in RIF patients

    doi: 10.1038/s41419-021-03679-8

    Figure Lengend Snippet: A hESCs were treated with Ad-LacZ or Ad-ATF3-His for 48 h and transfected with IRS-luc and Rinella for dual-luciferase assays. **** P < 0.0001. B – D hESCs were transfected with Ad-ATF3-his (0 or 40 MOI) or Ad-LacZ. si-FOXO1 was used to knockdown the expression of FOXO1, and prolactin secretion was measured by an enzyme-linked fluorescent assay (ELFA). * P < 0.05. Immunofluorescence was performed to analyze the morphological transformation of hESCs. Scale bar = 50 μm. E hESCs were transfected with Ad-ATF3-his (0, 20, or 40 MOI) or Ad-LacZ. FOXO1 and His protein expression levels were measured by western blot.

    Article Snippet: Immunoblotting was performed by incubating the membranes with primary antibodies against ATF3 (1:1000; HPA001562, Sigma), FOXO1 (1:2000; Cell Signaling Technology, Danvers, MA, United States), His-Tag (1:2000; M30111, Abmart, Shanghai, China), and GAPDH (1:10,000; AP0063, Bioworld Technology, MN, USA).

    Techniques: Transfection, Luciferase, Expressing, Fluorescence, Immunofluorescence, Transformation Assay, Western Blot

    A hESCs were transfected with Ad-ATF3-his (0 or 40 MOI) or Ad-LacZ. si-Dicer was used to knockdown the expression of Dicer, and prolactin secretion was measured by ELFA. * P < 0.05, ** P < 0.01, *** P < 0.001. B , C miRNA array and qPCR were performed to detect the differentially expressed miRNAs in RIF patients. Data were downloaded from the GEO database (GSE71332). The expression of miR-135b was validated by qPCR and normalized to U6 expression ( n = 9 for FER group and 12 for RIF group, * P < 0.05). D The correlation of ATF3 expression and miR-135b with the data in B . E hESCs were transfected with Ad-ATF3-his (0, 20, or 40 MOI) or Ad-LacZ. miR-135b expression levels were analyzed by qPCR. * P < 0.05. F GSEA results showed that the AAGCCAT miR-135B term was activated after overexpression of ATF3. G hESCs were treated with 8-Br-cAMP+MPA for different times, and then the expression of miR-135b was measured by qPCR. *** P < 0.001, **** P < 0.0001. H ChIP-PCR was used to detect the binding site of ATF3 on the promotor of miR-135b. I hESCs were transfected with miR-135b mimic (10 nM) or mimic control. After 24 h, the cells were treated with 0.5 mM 8-Br-cAMP and 1 μM MPA for an additional 3 days. Prolactin released into the medium was measured by ELFA. * P < 0.05, ** P < 0.01, *** P < 0.001. J hESCs were transfected with hsa-miR-135b mimic (0, 5, or 10 nM) or mimic control. FOXO1 protein levels were analyzed by western blot. ** P < 0.01. K , L Putative 7 bp paired miR-135b/a-target sites in the 3′UTR of human FOXO1 mRNA. Analysis of miR-135b modulation of the wild-type and mutant FOXO1 3′ UTR luciferase reporter activity. * P < 0.05, ns not significant.

    Journal: Cell Death & Disease

    Article Title: ATF3 deficiency impairs the proliferative–secretory phase transition and decidualization in RIF patients

    doi: 10.1038/s41419-021-03679-8

    Figure Lengend Snippet: A hESCs were transfected with Ad-ATF3-his (0 or 40 MOI) or Ad-LacZ. si-Dicer was used to knockdown the expression of Dicer, and prolactin secretion was measured by ELFA. * P < 0.05, ** P < 0.01, *** P < 0.001. B , C miRNA array and qPCR were performed to detect the differentially expressed miRNAs in RIF patients. Data were downloaded from the GEO database (GSE71332). The expression of miR-135b was validated by qPCR and normalized to U6 expression ( n = 9 for FER group and 12 for RIF group, * P < 0.05). D The correlation of ATF3 expression and miR-135b with the data in B . E hESCs were transfected with Ad-ATF3-his (0, 20, or 40 MOI) or Ad-LacZ. miR-135b expression levels were analyzed by qPCR. * P < 0.05. F GSEA results showed that the AAGCCAT miR-135B term was activated after overexpression of ATF3. G hESCs were treated with 8-Br-cAMP+MPA for different times, and then the expression of miR-135b was measured by qPCR. *** P < 0.001, **** P < 0.0001. H ChIP-PCR was used to detect the binding site of ATF3 on the promotor of miR-135b. I hESCs were transfected with miR-135b mimic (10 nM) or mimic control. After 24 h, the cells were treated with 0.5 mM 8-Br-cAMP and 1 μM MPA for an additional 3 days. Prolactin released into the medium was measured by ELFA. * P < 0.05, ** P < 0.01, *** P < 0.001. J hESCs were transfected with hsa-miR-135b mimic (0, 5, or 10 nM) or mimic control. FOXO1 protein levels were analyzed by western blot. ** P < 0.01. K , L Putative 7 bp paired miR-135b/a-target sites in the 3′UTR of human FOXO1 mRNA. Analysis of miR-135b modulation of the wild-type and mutant FOXO1 3′ UTR luciferase reporter activity. * P < 0.05, ns not significant.

    Article Snippet: Immunoblotting was performed by incubating the membranes with primary antibodies against ATF3 (1:1000; HPA001562, Sigma), FOXO1 (1:2000; Cell Signaling Technology, Danvers, MA, United States), His-Tag (1:2000; M30111, Abmart, Shanghai, China), and GAPDH (1:10,000; AP0063, Bioworld Technology, MN, USA).

    Techniques: Transfection, Expressing, Over Expression, Binding Assay, Western Blot, Mutagenesis, Luciferase, Activity Assay

    A GSEA results showed that the cell cycle term was activated in the endometrium of RIF patients (NES: 1.644476, P = 1.023820e−03). B . MKI67 staining was used to measure the proliferation of stromal cells in the endometrium of RIF patients and fertile controls (ST, stromal cells, GE, glandular epithelium, bar = 50 µm, n = 5). C immunofluorescence was performed to determine the expression of ATF3 and MKI67 in endometrium from fertile controls and RIF patients. D The cell cycle-regulated genes were measured by qPCR in the endometrium of RIF patients and fertile controls, n = 6. E TC-seq analysis was performed to analyze the DEGs in the control, decidualization, and siATF3+decidualization groups. Genes in clusters 1 and 6 were upregulated by siATF3, while those in cluster 2 were downregulated. F The correlation of ATF3 expression and CDKN1A. G hESCs were treated with Ad-ATF3-His or Ad-LacZ, and a CCK-8 assay was performed to detect the cell proliferation rate. H Genes related to cell proliferation and decidualization change regularly during in vitro-induced decidualization.

    Journal: Cell Death & Disease

    Article Title: ATF3 deficiency impairs the proliferative–secretory phase transition and decidualization in RIF patients

    doi: 10.1038/s41419-021-03679-8

    Figure Lengend Snippet: A GSEA results showed that the cell cycle term was activated in the endometrium of RIF patients (NES: 1.644476, P = 1.023820e−03). B . MKI67 staining was used to measure the proliferation of stromal cells in the endometrium of RIF patients and fertile controls (ST, stromal cells, GE, glandular epithelium, bar = 50 µm, n = 5). C immunofluorescence was performed to determine the expression of ATF3 and MKI67 in endometrium from fertile controls and RIF patients. D The cell cycle-regulated genes were measured by qPCR in the endometrium of RIF patients and fertile controls, n = 6. E TC-seq analysis was performed to analyze the DEGs in the control, decidualization, and siATF3+decidualization groups. Genes in clusters 1 and 6 were upregulated by siATF3, while those in cluster 2 were downregulated. F The correlation of ATF3 expression and CDKN1A. G hESCs were treated with Ad-ATF3-His or Ad-LacZ, and a CCK-8 assay was performed to detect the cell proliferation rate. H Genes related to cell proliferation and decidualization change regularly during in vitro-induced decidualization.

    Article Snippet: Immunoblotting was performed by incubating the membranes with primary antibodies against ATF3 (1:1000; HPA001562, Sigma), FOXO1 (1:2000; Cell Signaling Technology, Danvers, MA, United States), His-Tag (1:2000; M30111, Abmart, Shanghai, China), and GAPDH (1:10,000; AP0063, Bioworld Technology, MN, USA).

    Techniques: Staining, Immunofluorescence, Expressing, CCK-8 Assay, In Vitro

    A hESCs (from fertile controls and RIF patients) were treated with 8-Br-cAMP+MPA for 3 days. Prolactin secretion was measured by ELFA. n = 5, * P < 0.05, ** P < 0.01. B , C hESCs from RIF patients were infected with Ad-ATF3-his (0 or 40 MOI) or Ad-LacZ for 48 h followed by treatment with 0.5 mM 8-Br-cAMP and 1 μM MPA for 3 days. Prolactin released into the medium was measured by ELFA and qPCR. n = 4, * P < 0.05, ** P < 0.01. D Schematic representation of the role of ATF3 in the regulation of decidualization in RIF patients and fertile control groups.

    Journal: Cell Death & Disease

    Article Title: ATF3 deficiency impairs the proliferative–secretory phase transition and decidualization in RIF patients

    doi: 10.1038/s41419-021-03679-8

    Figure Lengend Snippet: A hESCs (from fertile controls and RIF patients) were treated with 8-Br-cAMP+MPA for 3 days. Prolactin secretion was measured by ELFA. n = 5, * P < 0.05, ** P < 0.01. B , C hESCs from RIF patients were infected with Ad-ATF3-his (0 or 40 MOI) or Ad-LacZ for 48 h followed by treatment with 0.5 mM 8-Br-cAMP and 1 μM MPA for 3 days. Prolactin released into the medium was measured by ELFA and qPCR. n = 4, * P < 0.05, ** P < 0.01. D Schematic representation of the role of ATF3 in the regulation of decidualization in RIF patients and fertile control groups.

    Article Snippet: Immunoblotting was performed by incubating the membranes with primary antibodies against ATF3 (1:1000; HPA001562, Sigma), FOXO1 (1:2000; Cell Signaling Technology, Danvers, MA, United States), His-Tag (1:2000; M30111, Abmart, Shanghai, China), and GAPDH (1:10,000; AP0063, Bioworld Technology, MN, USA).

    Techniques: Infection

    (A) Computational predicting binding sites of ATF3 and other transcription factors were shown in the up-stream of these genes. (B) Experimental results of ATF3 ChIP-seq and peaks for ATF3 binding sites were show in Integrative Genomics Viewer (IGV).

    Journal: Oncotarget

    Article Title: Overexpression of the transcription factor ATF3 with a regulatory molecular signature associates with the pathogenic development of colorectal cancer

    doi: 10.18632/oncotarget.16638

    Figure Lengend Snippet: (A) Computational predicting binding sites of ATF3 and other transcription factors were shown in the up-stream of these genes. (B) Experimental results of ATF3 ChIP-seq and peaks for ATF3 binding sites were show in Integrative Genomics Viewer (IGV).

    Article Snippet: Western blotting was performed using primary antibodies against ATF3 (1:500 dilution, Santa Cruz, USA) and actin (1: 10,000 dilution, Sigma, USA).

    Techniques: Binding Assay, ChIP-sequencing

    (A) Representative images of ATF3 expression in CRC cell lines: RKO and HCT116 expressed higher ATF3 protein level by Western blot analysis. (B) CEACAM1, DUSP14, HDC, HLF and ULBP2 mRNAexpressions were detected by qRT-PCR analysis. (C) RKO and HCT116 cells were transfected with ATF3 sgRNA, without doxycycline hyclate treatment as sgRNA CRISPR control, and with doxycycline hyclate treatment as CRISPR ATF3, which was confirmed by Western blot. (D, E) Silencing ATF3 caused changes in its target genes expressions by qRT-PCR analysis in HCT116 and RKO. Data represent mean ± SEM, (n=3). Paired t test in GraphPad Prism was used for data analysis. * represents p < 0.05. ** represents p < 0.01.

    Journal: Oncotarget

    Article Title: Overexpression of the transcription factor ATF3 with a regulatory molecular signature associates with the pathogenic development of colorectal cancer

    doi: 10.18632/oncotarget.16638

    Figure Lengend Snippet: (A) Representative images of ATF3 expression in CRC cell lines: RKO and HCT116 expressed higher ATF3 protein level by Western blot analysis. (B) CEACAM1, DUSP14, HDC, HLF and ULBP2 mRNAexpressions were detected by qRT-PCR analysis. (C) RKO and HCT116 cells were transfected with ATF3 sgRNA, without doxycycline hyclate treatment as sgRNA CRISPR control, and with doxycycline hyclate treatment as CRISPR ATF3, which was confirmed by Western blot. (D, E) Silencing ATF3 caused changes in its target genes expressions by qRT-PCR analysis in HCT116 and RKO. Data represent mean ± SEM, (n=3). Paired t test in GraphPad Prism was used for data analysis. * represents p < 0.05. ** represents p < 0.01.

    Article Snippet: Western blotting was performed using primary antibodies against ATF3 (1:500 dilution, Santa Cruz, USA) and actin (1: 10,000 dilution, Sigma, USA).

    Techniques: Expressing, Western Blot, Quantitative RT-PCR, Transfection, CRISPR, Control

    (A) Cell proliferation was determined by PrestoBlue Kit. (A-i) Significant difference was found in the numbers of RKO Mock cells, Control CRISPR cells and ATF3 CRISPR cells on day 4 and day 5. (A-ii) Significant difference also was found in the numbers of HCT116 Mock cells, Control CRISPR cells and ATF3 CRISPR cells on day 4 and day 5. (B) For wound healing assay, transfected cells were wounded by scratching and monitored over 24 hours to determine the rate of wound closure. Representative images of triplicate experiments are shown (B-i and B-iv) (10x). Cell migration of RKO and HCT116 cell lines were assessed by measuring relative wound closure (B-ii and B-v) and average speed (B-iii and B-vi) . (C) The metastatic properties of RKO and HCT116 cell lines and their transfected cells. Three independent experiments with three fields for each were performed and representative fields were shown (10x). Data represent mean ± SEM, (n=3). Paired t test in GraphPad Prism was used for data analysis. * represents p < 0.05. ** represents p < 0.01.

    Journal: Oncotarget

    Article Title: Overexpression of the transcription factor ATF3 with a regulatory molecular signature associates with the pathogenic development of colorectal cancer

    doi: 10.18632/oncotarget.16638

    Figure Lengend Snippet: (A) Cell proliferation was determined by PrestoBlue Kit. (A-i) Significant difference was found in the numbers of RKO Mock cells, Control CRISPR cells and ATF3 CRISPR cells on day 4 and day 5. (A-ii) Significant difference also was found in the numbers of HCT116 Mock cells, Control CRISPR cells and ATF3 CRISPR cells on day 4 and day 5. (B) For wound healing assay, transfected cells were wounded by scratching and monitored over 24 hours to determine the rate of wound closure. Representative images of triplicate experiments are shown (B-i and B-iv) (10x). Cell migration of RKO and HCT116 cell lines were assessed by measuring relative wound closure (B-ii and B-v) and average speed (B-iii and B-vi) . (C) The metastatic properties of RKO and HCT116 cell lines and their transfected cells. Three independent experiments with three fields for each were performed and representative fields were shown (10x). Data represent mean ± SEM, (n=3). Paired t test in GraphPad Prism was used for data analysis. * represents p < 0.05. ** represents p < 0.01.

    Article Snippet: Western blotting was performed using primary antibodies against ATF3 (1:500 dilution, Santa Cruz, USA) and actin (1: 10,000 dilution, Sigma, USA).

    Techniques: Control, CRISPR, Wound Healing Assay, Transfection, Migration

    (A) ATF3 immunohistochemical staining images of colon tissues from Wild type (WT) and ATF3 null (KO) mice (20x). (A-i) ATF3 staining scores of CRC tumor tissues and adjacent normal tissues from TMA. (A-ii) ATF3 staining scores of CRC tumor tissues in different clinical stages. Paired t test in GraphPad Prism was used for analysis in (B-i) and unpaired t test with Welch's correction in GraphPad Prism was used for analysis in (B-ii) . ** represents p < 0.01. (C) ATF3 staining images of different clinical stages (40x). (D) ATF3 staining scores of tumor tissues and adjacent normal tissues in different clinical stages. Paired t test in GraphPad Prism was used to compare the differences between the ATF3 staining scores of tumor tissues and adjacent normal tissues. ** represents p < 0.01.

    Journal: Oncotarget

    Article Title: Overexpression of the transcription factor ATF3 with a regulatory molecular signature associates with the pathogenic development of colorectal cancer

    doi: 10.18632/oncotarget.16638

    Figure Lengend Snippet: (A) ATF3 immunohistochemical staining images of colon tissues from Wild type (WT) and ATF3 null (KO) mice (20x). (A-i) ATF3 staining scores of CRC tumor tissues and adjacent normal tissues from TMA. (A-ii) ATF3 staining scores of CRC tumor tissues in different clinical stages. Paired t test in GraphPad Prism was used for analysis in (B-i) and unpaired t test with Welch's correction in GraphPad Prism was used for analysis in (B-ii) . ** represents p < 0.01. (C) ATF3 staining images of different clinical stages (40x). (D) ATF3 staining scores of tumor tissues and adjacent normal tissues in different clinical stages. Paired t test in GraphPad Prism was used to compare the differences between the ATF3 staining scores of tumor tissues and adjacent normal tissues. ** represents p < 0.01.

    Article Snippet: Western blotting was performed using primary antibodies against ATF3 (1:500 dilution, Santa Cruz, USA) and actin (1: 10,000 dilution, Sigma, USA).

    Techniques: Immunohistochemical staining, Staining

    Association between  ATF3  expression and clinicopathological characteristics in colon cancer patients

    Journal: Oncotarget

    Article Title: Overexpression of the transcription factor ATF3 with a regulatory molecular signature associates with the pathogenic development of colorectal cancer

    doi: 10.18632/oncotarget.16638

    Figure Lengend Snippet: Association between ATF3 expression and clinicopathological characteristics in colon cancer patients

    Article Snippet: Western blotting was performed using primary antibodies against ATF3 (1:500 dilution, Santa Cruz, USA) and actin (1: 10,000 dilution, Sigma, USA).

    Techniques: Expressing

    Univariate and multivariate analysis of prognostic factors for overall survival in colon cancer patients

    Journal: Oncotarget

    Article Title: Overexpression of the transcription factor ATF3 with a regulatory molecular signature associates with the pathogenic development of colorectal cancer

    doi: 10.18632/oncotarget.16638

    Figure Lengend Snippet: Univariate and multivariate analysis of prognostic factors for overall survival in colon cancer patients

    Article Snippet: Western blotting was performed using primary antibodies against ATF3 (1:500 dilution, Santa Cruz, USA) and actin (1: 10,000 dilution, Sigma, USA).

    Techniques: Expressing

    Overall survival curves by Kaplan–Meier method assessed for (A) ATF3 expression based on immunohistochemistry staining (p=0.007), (B) different clinical stages (p<0.001), (C) CEACAM1 mRNA expression (p=0.037), (D) DUSP14 mRNA expression (p=0.022), (E) HDC mRNA expression (p=0.015), (F) HLF mRNA expression (p=0.026), (G) ULBP2 mRNA expression (p=0.004). (A-B) were based on the results of immunohistochemistry. (C-G) were based on data from TCGA database.

    Journal: Oncotarget

    Article Title: Overexpression of the transcription factor ATF3 with a regulatory molecular signature associates with the pathogenic development of colorectal cancer

    doi: 10.18632/oncotarget.16638

    Figure Lengend Snippet: Overall survival curves by Kaplan–Meier method assessed for (A) ATF3 expression based on immunohistochemistry staining (p=0.007), (B) different clinical stages (p<0.001), (C) CEACAM1 mRNA expression (p=0.037), (D) DUSP14 mRNA expression (p=0.022), (E) HDC mRNA expression (p=0.015), (F) HLF mRNA expression (p=0.026), (G) ULBP2 mRNA expression (p=0.004). (A-B) were based on the results of immunohistochemistry. (C-G) were based on data from TCGA database.

    Article Snippet: Western blotting was performed using primary antibodies against ATF3 (1:500 dilution, Santa Cruz, USA) and actin (1: 10,000 dilution, Sigma, USA).

    Techniques: Expressing, Immunohistochemistry, Staining