Journal: Molecular cell

Article Title: Cockayne's Syndrome A and B Proteins Regulate Transcription Arrest after Genotoxic Stress by Promoting ATF3 Degradation.

doi: 10.1016/j.molcel.2017.11.009

Figure Lengend Snippet: Figure 1. Gene Expression in CSA, CSB, and Rescued Cells after Genotoxic Stress (A) Venn diagram of RNA-seq showing the number of downregulated genes upon UV irradiation in CSA- (CS3BE) and CSB- (CS1AN) deficient cells. (B) Amount of ATF3-dependent genes in common between CSA and CSB cells that were downregulated. (C) Pie chart showing genome-wide distribution of ATF3-binding events among the downregulated genes. (D) seqMINER heatmaps from ChIP-seq data showing site-specific ATF3 (lanes 1–4) and Pol II (lanes 5–8) binding events within 0, 8, and 24 hr after UV in CS1AN+CSBWT (WT) and CS1AN (Mut) cell lines. Data aligned to 8 hr ATF3 peak position. (E) RT-PCR of ATF3 in CS3BE, CS3BE+CSAWT, CS1AN, and CS1AN+CSBWT cells 4 hr after UV. (F) RT-PCR of DHFR, CDK5RAP2, NRG1, ID1, NIPBL, DYRK1A, and RAD50 genes in CS3BE, CS3BE+CSAWT, CS3BE+siATF3, and CS3BE+siCtrl cells, 24 hr after UV. Gene symbols are indicated at the right of each figure. Values are presented as fold expression in relation to the internal expression control GAPDH and to expression level of each gene at time point t = 0 (without UV irradiation). (G) Western blot of extracts from CS3BE cells transiently transfected with either siCtrl or siATF3; cells were collected 24 hr post-UV. Error bars represent SD from at least three independent experiments.

Article Snippet: Blots were incubated with antibody against ATF3 (Santa-Cruz, C-19), Biotin (Santa-Cruz, 39-15D9), Ubiquitin (Santa-Cruz, A-5).

Techniques: Gene Expression, RNA Sequencing, Irradiation, Genome Wide, Binding Assay, ChIP-sequencing, Reverse Transcription Polymerase Chain Reaction, Expressing, Control, Western Blot, Transfection

Journal: Molecular cell

Article Title: Cockayne's Syndrome A and B Proteins Regulate Transcription Arrest after Genotoxic Stress by Promoting ATF3 Degradation.

doi: 10.1016/j.molcel.2017.11.009

Figure Lengend Snippet: Figure 2. CSA and CSB Overcome ATF3-Dependent Transcriptional Repression (A1–D1) Western blot of ATF3 accumulation in (A1) CS3BE, (B1) CS3BE+CSAWT, (C1) CS1AN/CSBQ678E, and (D1) CS1AN/CSBQ678E+CSB WT cells over time (as indicated at the top of each panel) after UV irradiation; in the rescued cells, CSAWT and CSBWT were identified as well as a-tubulin as a control. (A2–D2) ChIP assay determining the presence of Pol II at the DHFR core promoter, and of ATF3, CSB, or CSBQ678E at the ATF/CRE site. (A3–D3) ChIP assay determining the presence of CSA, DDB1, and CUL4A and ChIP/reChIP assay for CSB/CSA. (A4–D4) ChIP/reChIP assay determining the presence of ATF3/CSB and CSB/Pol II at the CRE/ATF and core promoter of DHFR. (A5–D5) ChIP assay determining the presence of the acetylated H3K9, tri-methylated H3K4, or di-methylated H3K9 and tri-methylated H3K27 24 hr after UV. All results are presented as fold enrichment (ratio of the value obtained at each time point relative to time t = 0 in comparison with chromatin input). Each point represents the average of three RT-PCR reactions of three independent experiments. (E and F) Immunoprecipitation assays showing interaction between (E) ATF3 and CSB and CSA proteins in CS1AN+CSBWT whole-cell extract and (F) ATF3, CSA, and CUL4A in CS3BE+CSAWT whole-cell extract. (G and H) Reciprocal pull-down assays showing interactions between recombinant ATF3 and CSB proteins. Molecular weight is shown in kDa. Error bars represent SD from at least three independent experiments.

Article Snippet: Blots were incubated with antibody against ATF3 (Santa-Cruz, C-19), Biotin (Santa-Cruz, 39-15D9), Ubiquitin (Santa-Cruz, A-5).

Techniques: Western Blot, Irradiation, Control, Methylation, Comparison, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, Recombinant, Molecular Weight

Journal: Molecular cell

Article Title: Cockayne's Syndrome A and B Proteins Regulate Transcription Arrest after Genotoxic Stress by Promoting ATF3 Degradation.

doi: 10.1016/j.molcel.2017.11.009

Figure Lengend Snippet: Figure 3. CSA and CSB Are Promoting ATF3 Ubiquitination (A–D) ChIP/reChIP of Ubi/ATF3, Ubi/CSB, and CSB/MDM2 showing the presence of ATF3 and CSB in a ubiquitinated protein complex as well as the partnership between CSB and MDM2 at CRE/ATF site of DHFR. (E) Scheme of the setup and feature of the experiment. (F) Streptavidin immunoprecipitation assay from CS3BE+CSAWT, CS3BE, CS1AN+CSBWT, and CS1AN cells showing the ubiquitination of endogenous ATF3. Cells were treated or not with MG132 proteasomal inhibitor. Blot was performed against ATF3. (G) Streptavidin immunoprecipitation assay showing kinetic of endogenous ubiquitinated ATF3 in response to UV stress in CS3BE+CSAWT and CS3BE cells. Blot was performed against ATF3 (upper panel; middle panel represents a longer blot exposure) or against ubiquitin (lower panel). Time points, 0, 8, 16, and 24 hr; MG132 at 22 hr; cells were treated 2 hr with MG132; control (Co), no pEBB-BT-ubiquitin transfection. (H) In vitro ubiquitination of ATF3 in the presence of recombinant MDM2, CSA, and CSB as indicated at the top of the panel. Relative percentage of recombinant ATF3 ubiquitination is represented at the bottom of the panel. Error bars represent SD from at least three independent experiments.

Article Snippet: Blots were incubated with antibody against ATF3 (Santa-Cruz, C-19), Biotin (Santa-Cruz, 39-15D9), Ubiquitin (Santa-Cruz, A-5).

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Control, Transfection, In Vitro, Recombinant

Journal: Molecular cell

Article Title: Cockayne's Syndrome A and B Proteins Regulate Transcription Arrest after Genotoxic Stress by Promoting ATF3 Degradation.

doi: 10.1016/j.molcel.2017.11.009

Figure Lengend Snippet: Figure 4. CSA and CSB Promote Recruitment of the Proteasome for ATF3 Degradation (A1–D1) ChIP showing the recruitment of PSMD1 and PSMB5 subunits of proteasome to the ATF/CRE site of DHFR. (A2–D2) ChIP/reChIP showing the co-localization of CSB with either PSMD1 or PSMB5 at CRE/ATF site. (E) Heatmaps of ATF3, CSA, CSB, MDM2, and PSMB5 within 0 and 8 hr after UV irradiation; data aligned to 8 hr ATF3 peak position. (F) Site-specific peak distribution around ATF3 (intensity corresponds to the right axis) binding for CSA, CSB, and MDM2 (intensity corresponds to the left axis). (G and H) Immunoprecipitation assay showing the interaction between (G) CSA, PSMD1, and PSMB5 in CS3BE+CSAWT cells and (H) CSB, PSMD1, and PSMB5 in CS1AN+CSBWT cells. Both (G) and (H) are the parts of the original immunoprecipitations mentioned in Figures 2F and 2E, respectively. (I) In vitro degradation of ATF3 using whole-cell extracts from CS3BE, CS3BE + CSAWT, CS1AN, and CS1AN + CSBWT cells treated or not with UV and/or MG132 as indicated at the top of the panel. Incubation time was 6 hr. For each western blot, the resolution of the molecular weight is shown in kDa. Error bars represent SD from at least three independent experiments.

Article Snippet: Blots were incubated with antibody against ATF3 (Santa-Cruz, C-19), Biotin (Santa-Cruz, 39-15D9), Ubiquitin (Santa-Cruz, A-5).

Techniques: Irradiation, Binding Assay, Immunoprecipitation, In Vitro, Incubation, Western Blot, Molecular Weight

Journal: Molecular cell

Article Title: Cockayne's Syndrome A and B Proteins Regulate Transcription Arrest after Genotoxic Stress by Promoting ATF3 Degradation.

doi: 10.1016/j.molcel.2017.11.009

Figure Lengend Snippet: Figure 6. ATF3 Ubiquitination Is Crucial for RNA Synthesis Recovery (A) Fluorescence-activated cell sorting (FACS) analysis showing the rate of cell death upon UV irradiation in CS1AN cells transfected with siCtrl and siATF3. We assume the total apoptosis rate by counting events in upper left and right corners of histograms. (B) Western blot showing the levels of ATF3 in CS3BE and CS3BE KO ATF3 cells upon UV treatment as well as the efficiency of CUL4A and MDM2 protein silencing after respective siRNA treatment. (C1) ChIP assay determining the presence of CSB and Pol II at the DHFR core promoter as well as CSB/Pol II through ChIP/reChIP assay in CS3BE/KO ATF3 +CSAWT cells. (C2) ChIP assay determining the presence of CSA and CUL4A and ChIP/reChIP assay for CSB/CSA and CSB/MDM2 on CRE/ATF site. (C3) ChIP assay determining the presence of PSMD1 and PSMB5 proteasomal subunits on CRE/ATF site. (C4) ChIP/reChIP assay determining the presence of CSB/PSMD1 and CSB/PSMB5 interaction on CRE/ATF site of DHFR. (D) Reciprocal immunoprecipitation experiment showing the ubiquitination levels of WT, m1, and m2 mutant constructs of ATF3 co-transfected together with pEBB-BT-ubiquitin plasmid into 293T cells. Streptavidin precipitation (left panel) or vice versa anti-flag immunoprecipitation (right panel) revealed with anti-ATF3 antibody or with anti-biotin antibody, detecting the biotin-ubiquitinated fraction. Control represents cells transfected only with pEBB-BT-ubiquitin construct. The pattern shows the ubiquitinated form of ATF3. (E) FACS analysis showing the rate of cell death upon UV irradiation in CS1AN cells transfected with WT, m1, and m2 ATF3 constructs. The percentage from three independent experiments was presented in the histogram in Figure S1E.

Article Snippet: Blots were incubated with antibody against ATF3 (Santa-Cruz, C-19), Biotin (Santa-Cruz, 39-15D9), Ubiquitin (Santa-Cruz, A-5).

Techniques: Ubiquitin Proteomics, Fluorescence, FACS, Irradiation, Transfection, Western Blot, Immunoprecipitation, Mutagenesis, Construct, Plasmid Preparation, Control

Journal: bioRxiv

Article Title: ATF3 Preserves Skeletal Muscle Stem Cell Quiescence by Preventing Precocious Activation

doi: 10.1101/2022.08.12.503549

Figure Lengend Snippet: A Schematic for isolation of quiescent satellite cells (QSC) after in situ fixation, freshly isolated SCs (FISC) without prior fixation from muscles of Tg: Pax7-nGFP mice. FISCs were subsequently cultured and activated for 24 (ASC-24h), 48 (ASC-48h), or 72 h (DSC-72h). RNAs were extracted for RNA-Seq analysis. B Heat maps indicating gene expression levels (Log2[FPKM]) of AP-1 family TFs detected by the RNA-Seq. C-D Expression levels (FPKM) and genomic snapshots of Atf3 , Atf4 , Fos , FosB , JunB mRNAs from the above RNA-Seq. E RT-qPCR detection of Atf3 in the above cells. F Immunofluorescence (IF) staining of ATF3 and Pax7 protein on the above cells. Scale bar: 50 μm. G IF staining of ATF3 and Pax7 protein on single myofibers from EDL muscles immediately after isolation or cultured for 24h. Scale bar: 25 μm. All the bar graphs are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. n.s., no significance.

Article Snippet: 10 μg of antibodies against ATF3 (Santa Cruz Biotechnology), or normal mouse IgG (Santa Cruz Biotechnology) was used for immunoprecipitation.

Techniques: Isolation, In Situ, Muscles, Cell Culture, RNA Sequencing, Gene Expression, Expressing, Quantitative RT-PCR, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: ATF3 Preserves Skeletal Muscle Stem Cell Quiescence by Preventing Precocious Activation

doi: 10.1101/2022.08.12.503549

Figure Lengend Snippet: A Breeding scheme for generating inducible Atf3 inducible conditional knock out ( Atf3 iKO), Pax7 CreER/+ ; ROSA EYFP/+ ; Atf3 fl/fl , and control (Ctrl), Pax7 CreER/+ ; ROSA EYFP/+ ; Atf3+/+ , littermates. B Schematic outline of the tamoxifen (TMX) administration used in the study and experimental design for testing the effect of short-term Atf3 deletion on barium chloride (BaCl 2 ) induced muscle regeneration process. C Loss of ATF3 protein in iKO FISC was confirmed by Western blot. α -Tubulin was used as a loading control. D No overt morphological difference or body weight change was observed in representative Ctrl vs iKO mice. E Left: H&E staining of the TA muscles collected at 3, 5 and 7 days post the 1 st round injury (dpi). Scale bar: 50 μm. Right: Cross-sectional areas (CSAs) of newly formed fibers were quantified from the TA muscles at 7dpi and the distribution is shown. n = 3 mice per group. F Left: IF staining of eMyHC (red) and Laminin (green) was performed on the above TA muscles. Scale bar: 50 μm. Right: the numbers of eMyHC+ fibers per view at 3dpi were quantified. n = 3 mice per group. G-H Left: H&E and eMyHC staining of the TA muscles collected at 3, 5 and 7 days post the 2 nd round injury. Scale bar: 50 μm. n = 3 mice per group. I-J H&E and eMyHC staining of the TA muscles collected at 3, 5 and 7 days post the 3 rd round injury. Scale bar: 50 μm. n = 3 mice per group. K Left: IF staining of Pax7 (red) and Laminin (green) on Ctrl and iKO TA muscles on 30 days post the 3 rd round of injury. Scale bar: 50 μm. Right: the numbers of Pax7+ SCs per view were quantified. n = 3 mice per group. L Schematic for engraftment. FISCs from donor mice (Ctrl/ iKO) were injected into pre-injured (1 day before) receptor nude mice. TA muscles were collected 21 days after engraftment for analysis. M Left: IF staining of YFP (green) and Laminin (red) on the TA muscles 21 days after engraftment. Scale bar: 50 μm. Right: the numbers of YFP+ fibers per view at 21 days after engraftment were quantified. n= 3 mice per group. N IF staining of Myod (red) and Laminin (green) on Ctrl and iKO TA muscles at 3 and 5 days post the 1 st , 2 nd and 3 rd rounds of injury. Scale bar: 50 μm; n = 3 mice per group. O Quantification of the numbers of Myod+ SCs per view on the above TA muscles (N). n = 3 mice per group. All the bar graphs are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. n.s., no significance.

Article Snippet: 10 μg of antibodies against ATF3 (Santa Cruz Biotechnology), or normal mouse IgG (Santa Cruz Biotechnology) was used for immunoprecipitation.

Techniques: Knock-Out, Control, Western Blot, Staining, Muscles, Injection

Journal: bioRxiv

Article Title: ATF3 Preserves Skeletal Muscle Stem Cell Quiescence by Preventing Precocious Activation

doi: 10.1101/2022.08.12.503549

Figure Lengend Snippet: A Schematic outline of the experimental design for testing the effect of long-term Atf3 deletion on muscle regeneration process. A 30 or 120 days chasing after TMX injection was given before BaCl 2 injection. B Left: H&E staining of the TA muscles collected at 3, 5 and 7 dpi after the 30 days chasing period. Scale bar: 50 μm. n = 3 mice per group. Right: CSAs of newly formed fibers were quantified from the above-stained TA muscle at 7dpi and the distribution is shown. n = 3 mice per group. C Left: IF staining of eMyHC (red) and Laminin (green) was performed on the above TA muscles. Scale bar: 50 μm. Right: Quantification of the numbers of eMyHC+ fibers per view at 5 and 7dpi. n = 3 mice per group. D Left: IF staining of Pax7 (red) and Laminin (green) was performed on the above TA muscles. Scale bar: 50 μm. Right: Quantification of the numbers of Pax7+ SCs per view. n = 3 mice per group. E-G The above assays/quantifications were performed on the Ctrl or iKO muscles collected at 3, 5 and 7 dpi after the 120 days chasing period. Scale bar: 50 μm. n = 3 mice per group. All the bar graphs are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. n.s., no significance.

Article Snippet: 10 μg of antibodies against ATF3 (Santa Cruz Biotechnology), or normal mouse IgG (Santa Cruz Biotechnology) was used for immunoprecipitation.

Techniques: Injection, Staining, Muscles

Journal: bioRxiv

Article Title: ATF3 Preserves Skeletal Muscle Stem Cell Quiescence by Preventing Precocious Activation

doi: 10.1101/2022.08.12.503549

Figure Lengend Snippet: A Schematic outline of the experimental design. RNA-Seq and H2B CUT&RUN were performed with FISCs isolated from Ctrl or iKO mice; ATF3 ChIP-Seq was performed with C2C12 overexpressing ATF3. The acquired datasets were then integrated for analyses. B Left: Differentially expressed genes (DEGs) were identified from the above Ctrl vs. iKO RNA-Seq. Right: The percentages of up and down-regulated genes. C&D Gene ontology (GO) analyses of the above up and down-regulated genes. The top 5 enriched GO terms ranked by gene ratio (proportion of genes annotated for each GO term) are shown. Dots are colored by adjusted P value of fold change (FC) and their size corresponds to the gene counts annotated to each GO term. E FPKM and Log2FC of H2b genes in iKO vs. Ctrl from the above RNA-Seq. F ChIP-Seq was performed using chromatins from C2C12 cells overexpressing ATF3, and the genomic distribution of 2871 ATF3 binding peaks is shown. G De novo motif prediction by DREME revealed the enrichment of canonical ATF3 motifs in the above binding regions. H The GO analysis for all the above genes with ATF3 binding. The top ten enriched GO terms ranked by gene ratio are shown. Dots are colored by adjusted P value (degree of enrichment) and their size corresponds to the gene counts annotated to each GO term. I Left: Venn diagrams showing the overlapping (72 genes) between the above identified ChIP-Seq target (2871) and the down-regulated genes (469) from the RNA-Seq. Right: GO analysis of the above 72 genes revealed an extreme enrichment of histone-related terms. J Genomic snapshots of 3 of the above identified H2b genes ( Hist1h2bb , Hist1h2bm and Hist2h2bb ) with ATF3 binding in their TSSs (ChIP-Seq tracks) and down-regulated by ATF3 deletion (RNA-Seq tracks). K ATF3, H2A, H2B, H3 and H4 proteins were measured by western blot in FISC from Ctrl and iKO mice. α-Tubulin was used as a loading control. L IF staining of H2A (green) and H2B (red) on the above FISCs. Scale bar: 100 μm; n = 3 mice per group. M H2B CUT&RUN was performed with FISCs isolated from Ctrl or iKO mice. The enrichment level in iKO vs. Ctrl was compared by analyzing gains and losses of H2B reads (fold change 1.5) in 10 kb bins. Pie charts showing the number of the bins with H2B changed (6589, 229 up and 6369 down) and unchanged (247114) (left and middle), and the genomic distribution of the down-regulated H2B signals (right). N Genomic snapshots of a 1098kb region Integrative Genomics Viewer (IGV) track of H2B signal in-Ctrl vs. iKO in Chr4. O Schematic of the RNA Polymerase II elongation process during DNA transcription P Venn diagrams showing the overlapping (252 genes) between the above regions with down-regulated H2B CUT&RUN signals (4333) and the up-regulated genes (1866) from the above (A) RNA-Seq. Q Metaplots showing average H2B CUT&RUN signals in Ctrl and iKO cell 3 kb upstream to 3 kb downstream of TSS. R GO analysis of the above 252 genes from (P). The top ten enriched GO terms ranked by gene ratio are shown. Dots are colored by adjusted P value (degree of enrichment) and their size corresponds to the gene counts annotated to each GO term. S Genomic snapshots of Eln gene which shows down-regulated H2B CUT&RUN signal and up-regulated RNA-Seq signal in iKO vs. Ctrl. Regions with H2B signal reduction in iKO vs. Ctrl are highlighted in blue frame.

Article Snippet: 10 μg of antibodies against ATF3 (Santa Cruz Biotechnology), or normal mouse IgG (Santa Cruz Biotechnology) was used for immunoprecipitation.

Techniques: RNA Sequencing, Isolation, ChIP-sequencing, Binding Assay, Western Blot, Control, Staining

Journal: bioRxiv

Article Title: ATF3 Preserves Skeletal Muscle Stem Cell Quiescence by Preventing Precocious Activation

doi: 10.1101/2022.08.12.503549

Figure Lengend Snippet: A Upper: Comet assay was performed on Ctrl or iKO SCs after 0, 1000 or 2000J UV treatment. Scale bar: 25 μm. Lower: Quantification of the length of comet tails; n = 3 mice per group. B IF staining of γH2AX (red) and Myod (green) was performed on SCs from Ctrl or iKO mice after cultured for 0, 24, 48 or 72hours. Scale bar: 50 μm. C Quantification of the percentages of Myod+ / γH2AX+ cells; n = 3 mice per group. D Left: β-Gal (blue) staining was performed on SCs from Ctrl or iKO mice after cultured for 9 days. Scale bar: 50 μm. Right: Quantification of the percentages of β-Gal+ cells; n = 3 mice per group. E Expression of the selected senescence marker genes including p16 , p21 and p53 in SCs from Ctrl and iKO was quantified by RT– qPCR . F p21 and p53 protein levels were detected by Western blotting in SCs from Ctrl and iKO mice. GAPDH was used as a loading control. G&H SCs from Ctrl and iKO mice were transfected with an H2B-overexpression or vector control plasmid. 96hours after transfection, expression of the indicated genes was detected by Western blotting and RT–qPCR. GAPDH was used as a loading control. I Left: The above transfected cells were cultured for 24 h before treated with EdU for 6 h and stained for EdU (red) and Pax7 (green). Scale bar: 50 μm. Right: Quantification of the percentage of EdU+/Pax7+ SCs; n = 3 mice per group. J Schematic model depicting the functional role of ATF3 in preserving SC quiescence and preventing SC precocious activation. In homeostatic muscle, the rapid induction of ATF3 upon minor stress promotes H2b expression to maintain proper nucleosome positioning and suppress the expression of activation genes, thus preventing the precocious activation of SCs. With ATF3 loss, H2B deficiency occurs which leads to altered nucleosomes positioning and the up-regulation of activation genes, causing SCs to break quiescence (QSC) and enter early activating stage (eASC). All the bar graphs are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. n.s., no significance.

Article Snippet: 10 μg of antibodies against ATF3 (Santa Cruz Biotechnology), or normal mouse IgG (Santa Cruz Biotechnology) was used for immunoprecipitation.

Techniques: Single Cell Gel Electrophoresis, Staining, Cell Culture, Expressing, Marker, Quantitative RT-PCR, Western Blot, Control, Transfection, Over Expression, Plasmid Preparation, Functional Assay, Preserving, Activation Assay

Journal: iScience

Article Title: Endothelial activating transcription factor 3 promotes angiogenesis and vascular repair in the mouse retina

doi: 10.1016/j.isci.2024.111516

Figure Lengend Snippet:

Article Snippet: The primary antibodies were anti-ATF3 (1/1000, Cell Signaling Technology) and anti-βactin (1/5000, Sigma-Aldrich).

Techniques: Recombinant, Transfection, Isolation, Gene Expression, Negative Control, Software

Journal: Discover Oncology

Article Title: Comprehensive analysis of ATF3 as a diagnostic and prognostic biomarker from pan-cancer to clear cell renal cell carcinoma

doi: 10.1007/s12672-026-05113-x

Figure Lengend Snippet: Expression and diagnostic significance of ATF3 in pan-cancer. A ATF3 expression in normal and cancer samples analyzed with the GTEx and TCGA datasets. B Receiver operating characteristic (ROC) analysis evaluated the diagnostic performance of ATF3 across cancer types. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001

Article Snippet: After 30 min of blocking with 5% non-fat milk, the membranes were incubated for the whole night at 4 °C with primary antibodies against ATF3 (1:500, ABclonal) and GAPDH (1:10,000, ABclonal).

Techniques: Expressing, Diagnostic Assay

Journal: Discover Oncology

Article Title: Comprehensive analysis of ATF3 as a diagnostic and prognostic biomarker from pan-cancer to clear cell renal cell carcinoma

doi: 10.1007/s12672-026-05113-x

Figure Lengend Snippet: Prognostic evaluation of ATF3 in pan-cancer. A – D Univariate Cox regression analyses of ATF3 in different tumors for disease-free interval (DFI) ( A ), disease-specific survival (DSS) ( B ), overall survival (OS) ( C ) and progression-free interval (PFI) ( D ). E – H Patients were stratified by the median ATF3 expression; Kaplan-Meier curves compared the impact of high vs. low ATF3 expression on DFI ( E ), DSS ( F ), OS ( G ) and PFI ( H ) in each cancer type. HR, hazard ratio; CI, confidence interval

Article Snippet: After 30 min of blocking with 5% non-fat milk, the membranes were incubated for the whole night at 4 °C with primary antibodies against ATF3 (1:500, ABclonal) and GAPDH (1:10,000, ABclonal).

Techniques: Expressing

Journal: Discover Oncology

Article Title: Comprehensive analysis of ATF3 as a diagnostic and prognostic biomarker from pan-cancer to clear cell renal cell carcinoma

doi: 10.1007/s12672-026-05113-x

Figure Lengend Snippet: Mutation landscape and mutation-load analysis of ATF3 in pan-cancer. A Frequencies of distinct ATF3 mutation types across cancers. B Analysis showing that missense mutations (green) were the predominant ATF3 alteration. C Radar plot depicting the correlation between ATF3 expression and tumor mutation burden (TMB). D Radar plot depicting the correlation between ATF3 expression and microsatellite instability (MSI). *, P < 0.05; **, P < 0.01

Article Snippet: After 30 min of blocking with 5% non-fat milk, the membranes were incubated for the whole night at 4 °C with primary antibodies against ATF3 (1:500, ABclonal) and GAPDH (1:10,000, ABclonal).

Techniques: Mutagenesis, Expressing

Journal: Discover Oncology

Article Title: Comprehensive analysis of ATF3 as a diagnostic and prognostic biomarker from pan-cancer to clear cell renal cell carcinoma

doi: 10.1007/s12672-026-05113-x

Figure Lengend Snippet: Relationship between ATF3 expression and immunity/differentially methylated probes-based stemness index (DMPsi). A ESTIMATE analysis of the association between ATF3 expression and ImmuneScore, StromalScore, and EstimateScore. B – F Correlations of ATF3 expression with immune-checkpoint genes ( B ), chemokines (C), immune inhibitors (D), immune stimulators (E) and chemokines receptors (F). G Correlations between ATF3 and mismatch-repair and DNA-methyltransferase genes. H Correlation between ATF3 and DMPsi. *, P < 0.05; **, P < 0.01; ***, P < 0.001

Article Snippet: After 30 min of blocking with 5% non-fat milk, the membranes were incubated for the whole night at 4 °C with primary antibodies against ATF3 (1:500, ABclonal) and GAPDH (1:10,000, ABclonal).

Techniques: Expressing, Methylation

Journal: Discover Oncology

Article Title: Comprehensive analysis of ATF3 as a diagnostic and prognostic biomarker from pan-cancer to clear cell renal cell carcinoma

doi: 10.1007/s12672-026-05113-x

Figure Lengend Snippet: ATF3 was down-regulated in kidney clear cell renal cell carcinoma (KIRC) and associated with favorable prognosis. A – C ATF3 expression in KIRC cell lines determined by real-time quantitative polymerase chain reaction (RT-qPCR) ( A ), Western blot ( B ) and immunofluorescence ( C ). D Kaplan-Meier survival curves comparing high vs. low ATF3 expression for DFI, DSS, OS and PFI in KIRC. E Association between ATF3 and clinical-feature subgroups in KIRC. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001

Article Snippet: After 30 min of blocking with 5% non-fat milk, the membranes were incubated for the whole night at 4 °C with primary antibodies against ATF3 (1:500, ABclonal) and GAPDH (1:10,000, ABclonal).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Immunofluorescence

Journal: Discover Oncology

Article Title: Comprehensive analysis of ATF3 as a diagnostic and prognostic biomarker from pan-cancer to clear cell renal cell carcinoma

doi: 10.1007/s12672-026-05113-x

Figure Lengend Snippet: Enrichment and immune-infiltration analyses of ATF3. A Volcano plot of differentially expressed genes (DEGs) between high and low ATF3 groups. B Heat-map of the DEGs. C Gene Ontology (GO) enrichment of the DEGs. BP, Biological Process; CC, Cellular Component; MF, Molecular Function. D Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment of the DEGs. E Gene set enrichment analysis (GSEA) of ATF3. F Stacked bar chart of immune-cell scores in high and low ATF3 groups. G Box plot showing differences in immune-cell infiltration between the two groups. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001

Article Snippet: After 30 min of blocking with 5% non-fat milk, the membranes were incubated for the whole night at 4 °C with primary antibodies against ATF3 (1:500, ABclonal) and GAPDH (1:10,000, ABclonal).

Techniques:

Journal: Discover Oncology

Article Title: Comprehensive analysis of ATF3 as a diagnostic and prognostic biomarker from pan-cancer to clear cell renal cell carcinoma

doi: 10.1007/s12672-026-05113-x

Figure Lengend Snippet: Prediction of ATF3-targeted drugs. A Drug-sensitivity analysis between high and low ATF3 groups (top 10 agents). B ATF3-drug network. C Molecular-docking affinity between progesterone and ATF3

Article Snippet: After 30 min of blocking with 5% non-fat milk, the membranes were incubated for the whole night at 4 °C with primary antibodies against ATF3 (1:500, ABclonal) and GAPDH (1:10,000, ABclonal).

Techniques:

Journal: Discover Oncology

Article Title: Comprehensive analysis of ATF3 as a diagnostic and prognostic biomarker from pan-cancer to clear cell renal cell carcinoma

doi: 10.1007/s12672-026-05113-x

Figure Lengend Snippet: Single-cell RNA sequencing (scRNA-seq) analysis of ATF3 in KIRC. A Uniform Manifold Approximation and Projection (UMAP) showing cell annotations. B Proportions of annotated cells in KIRC vs. control. C ATF3 expression across cell types in KIRC vs. control. D Pseudotime trajectory of endothelial-afferent/efferent arterioles/descending vasa recta (AEAs/DVR) divided into three stages. E Distribution of KIRC and control samples along the AEAs/DVR differentiation trajectory. F ATF3 expression during AEAs/DVR differentiation. G Cell-cell communication analysis. *, P < 0.05; **, P < 0.01; ****, P < 0.0001

Article Snippet: After 30 min of blocking with 5% non-fat milk, the membranes were incubated for the whole night at 4 °C with primary antibodies against ATF3 (1:500, ABclonal) and GAPDH (1:10,000, ABclonal).

Techniques: Single Cell, RNA Sequencing, Control, Expressing

Journal: Scientific Reports

Article Title: Usp9X Regulates Cell Death in Malignant Peripheral Nerve Sheath Tumors

doi: 10.1038/s41598-018-35806-5

Figure Lengend Snippet: Usp9X inhibition causes Noxa increase and ER stress in MPNST cell lines. Ultrastructural analysis shows features of paraptosis. ( a , b ) ST88-14 cells were transfected for 24 h with either non-targeting (NT)-siRNA or Usp9X-siRNA ( a ) or treated with WP1130 at the concentration of 1.25 and 2.5 µM ( b ). Whole cell extracts were collected prior to Western blot analysis for ATF3, Noxa and ß-actin. Numbers shows protein quantification analyzed through ImageJ. N = 3. ( c – e ) Ultrastructural appearance of untreated control cells using TEM. ( f – h ) After treatment with WP1130 at the concentration of 2.5 µM ( f , g , h ) T265-2c cells showed extensive cytosolic vacuolization (f, red arrows) and swelling of ER (g, red arrowheads) and mitochondria (h, red arrows).

Article Snippet: Primary antibodies were obtained from the following sources: Usp9X (Cell Signaling, Danvers, MA #5751), Mcl-1 (Cell Signaling, Danvers, MA #5453), Noxa (Calbiochem, San Diego, CA #OP180), ATF4 (Cell Signaling, Danvers, MA #11815), ATF3 (Novus Biologicals, Littleton, CO #NBP1-85816), β-actin (Sigma, St. Louis, MO #A5316).

Techniques: Inhibition, Transfection, Concentration Assay, Western Blot, Control

Journal: Journal of Cell Science

Article Title: Key role for Rac in the early transcriptional response to extracellular matrix stiffness and stiffness-dependent repression of ATF3

doi: 10.1242/jcs.260636

Figure Lengend Snippet: Rac is dominant over Rho in the initial transcriptome-wide response to ECM stiffness and preferentially represses ATF3. (A,B) Venn diagrams of differentially expressed genes in MEFs cultured with 10% FBS for 1 h on stiff versus soft hydrogels, stiff hydrogels with or without EHT1864, or stiff hydrogels with or without CT04. (C,D) The genes regulated by ECM and Rac in A,B were compared to GO gene lists for transcription factors (TFs), transcription co-regulators (co-reg) and histone modifiers. (E,F) Log 2 (fold change) values and adjusted P -values of the genes regulated by ECM stiffness and Rac and contained within the indicated GO terms above. (G) Serum-starved MEFs were plated on soft or stiff FN-coated hydrogels with 10% FBS for 1 h with DMSO (Ctrl), EHT1864 or CT04. Atf3 mRNA levels were quantified by RT-qPCR. The graph shows mean+s.e.m. ( n =4) with results normalized to the expression level on soft hydrogels. * P <0.05; *** P <0.001 (two-tailed unpaired t -tests).

Article Snippet: The membranes were saturated with 5% BSA in 1× TBS (20 mM Tris-HCl, pH 7.5, 150 mM NaCl) with 0.1% Tween-20 and probed with primary antibodies to ATF3 (1:200, sc-188, Santa Cruz Biotechnology or 1:500, NBP1-85816, Novus Biologicals), cyclin D1 (1:200, sc-20044, Santa Cruz Biotechnology or 1:500, 681902, BioLegend), Rac1/2/3 (1:100, 2465, Cell Signaling Technology), RhoA/B/C (1:200, MA1-011, Thermo Fisher Scientific) or GAPDH (loading control; 1:1000, MA5-15738, Thermo Fisher Scientific).

Techniques: Cell Culture, Quantitative RT-PCR, Expressing, Two Tailed Test

Journal: Journal of Cell Science

Article Title: Key role for Rac in the early transcriptional response to extracellular matrix stiffness and stiffness-dependent repression of ATF3

doi: 10.1242/jcs.260636

Figure Lengend Snippet: Dose-dependent effects of ECM stiffness on Rac–GTP, Atf3 mRNA, cyclin D1 mRNA and S phase entry. Serum-starved MEFs were incubated in DMEM containing 10% FBS on FN-coated hydrogels of increasing stiffness (∼2, 8, 15 and 25 kPa). (A) Rac–GTP levels determined at 1 h and graphed relative to Rac activity on the softest hydrogel. Results show mean±s.e.m. ( n =4). (B,C) Atf3 and cyclin D1 mRNA levels determined after 9 h and graphed relative to the mRNA levels on the softest hydrogel. Results show mean±s.e.m. ( n =3). (D) The percentage of EdU-positive nuclei determined at 24 h and graphed relative to EdU incorporation on the softest hydrogel. Results show mean±s.d. ( n =3). Statistical significance for each panel was determined by one-way ANOVA; asterisks show the results of Dunnett's post-tests relative to the softest hydrogel. * P <0.05; *** P <0.001; **** P <0.0001.

Article Snippet: The membranes were saturated with 5% BSA in 1× TBS (20 mM Tris-HCl, pH 7.5, 150 mM NaCl) with 0.1% Tween-20 and probed with primary antibodies to ATF3 (1:200, sc-188, Santa Cruz Biotechnology or 1:500, NBP1-85816, Novus Biologicals), cyclin D1 (1:200, sc-20044, Santa Cruz Biotechnology or 1:500, 681902, BioLegend), Rac1/2/3 (1:100, 2465, Cell Signaling Technology), RhoA/B/C (1:200, MA1-011, Thermo Fisher Scientific) or GAPDH (loading control; 1:1000, MA5-15738, Thermo Fisher Scientific).

Techniques: Incubation, Activity Assay

Journal: Journal of Cell Science

Article Title: Key role for Rac in the early transcriptional response to extracellular matrix stiffness and stiffness-dependent repression of ATF3

doi: 10.1242/jcs.260636

Figure Lengend Snippet: ATF3 repression linked to stiffness-dependent cyclin D1 expression. (A) Serum-starved MEFs on soft or stiff FN-coated hydrogels in DMEM containing 10% FBS were treated with vehicle (DMSO) or EHT1864 for 9 h. Atf3 and cyclin D1 mRNA levels were determined from the same lysates and normalized to mRNA expression levels in cells on soft hydrogels. Results show mean±s.e.m. ( n =3). (B) MEFs were infected with adenoviruses encoding GFP (control) or Rac V12 , serum-starved, and cultured and analyzed as in panel A. Results show mean±s.e.m. ( n =3). (C) MEFs infected with adenoviruses (Ad) encoding GFP (control) or ATF3 were serum-starved, incubated on FN-coated hydrogels with 10% FBS for 15 h, and analyzed by immunoblotting for cyclin D1 and ATF3 with GAPDH as the loading control. (D) Quantification of the immunoblot results in C. The graph shows mean+s.d. with results normalized to GAPDH abundance and plotted relative to the normalized cyclin D1 signal on the soft hydrogels ( n =3). (E,F) Serum-starved ROSA and ATF3 KO MEFs were incubated on stiff FN-coated hydrogels with DMSO (Ctrl) or EHT1864 for 9 h. Lysates were analyzed for the levels of ATF3 and cyclin D1 by immunoblotting. GAPDH was used as the loading control. Panel E shows results from ROSA clone R12 and ATF3 KO clone 1-20, and panel F shows quantification of the combined results from ROSA clones R11, R12 and R15 and ATF3 KO clones 1-20 and 1-29. Data were accrued from four independent experiments, and the graph shows mean+s.d. with results normalized to GAPDH abundance and plotted relative to the normalized cyclin D1 signal in the ROSA control. (G) S phase entry was analyzed by EdU incorporation in ROSA clones (R3, R11, R12 and R15) and ATF3 KO clones (1-20, 1-29, 1-44, 1-48 and 1-49) after serum starvation and incubation on stiff FN-coated hydrogels in DMEM containing 10% FBS for 24 h with DMSO (Ctrl) or EHT1864. Results show mean+s.d. n =7 for the ROSA clones and n =8 for the ATF3 KO clones. (H) Model showing that cyclin D1 is regulated by ECM stiffness and Rac through ATF3. * P <0.05; ** P <0.01; **** P <0.0001 (D, two-tailed unpaired t -tests; F and G, one-tailed unpaired t -tests).

Article Snippet: The membranes were saturated with 5% BSA in 1× TBS (20 mM Tris-HCl, pH 7.5, 150 mM NaCl) with 0.1% Tween-20 and probed with primary antibodies to ATF3 (1:200, sc-188, Santa Cruz Biotechnology or 1:500, NBP1-85816, Novus Biologicals), cyclin D1 (1:200, sc-20044, Santa Cruz Biotechnology or 1:500, 681902, BioLegend), Rac1/2/3 (1:100, 2465, Cell Signaling Technology), RhoA/B/C (1:200, MA1-011, Thermo Fisher Scientific) or GAPDH (loading control; 1:1000, MA5-15738, Thermo Fisher Scientific).

Techniques: Expressing, Infection, Control, Cell Culture, Incubation, Western Blot, Clone Assay, Two Tailed Test, One-tailed Test

Journal: Experimental & Molecular Medicine

Article Title: Decreased expression of ATF3, orchestrated by β-catenin/TCF3, miR-17-5p and HOXA11-AS, promoted gastric cancer progression via increased β-catenin and CEMIP

doi: 10.1038/s12276-021-00694-9

Figure Lengend Snippet: a ATF3 expression data as measured by RNA-seq or a normalized probe were retrieved from The Cancer Genome Atlas (TCGA STAD, i) and Gene Expression Omnibus (GEO, GSE27342-ii, GSE66229-iii) databases, respectively, and were used for comparison between GC samples and normal tissues. The data are presented as a scatter plot. b ATF3 mRNA expression was compared between 46 fresh GC samples and their paired adjacent tissues. c The expression of ATF3 mRNA (i) and protein (ii) was detected by RT-qPCR and western blot, respectively, in nine GC cell lines. d IHC scores of 90 pairs of GC tissues in the TMA180Su09 dataset based on ATF3 expression analyzed by IHC staining. e Representative IHC staining results of ATF3 in two human GC tissues and their paired normal tissues (scale bar, 20 μm). f Kaplan–Meier (K-M) analysis of overall survival (OS) of 90 GC patients according to the ATF3 IHC scores (data from the TMA180Su09 dataset). g i) K–M plot of OS of 876 GCs; ii) K–M plot of first progression survival (FPS) of 641 GCs; iii) K–M plot of post progression survival (PPS) of 499 GCs. The data were obtained from an online dataset from K–M Plotter with 202672_s_as the probe, and GC patients were stratified by the median ATF3 expression ( p = 0.0049 for OS; p = 6.4e-05 for FP; p = 0.0002 for PPS; log-rank test). * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student’s t test compared with the normal group.

Article Snippet: Western blots were performed as described with primary antibodies targeting ATF3 (1:1000, #33593, Cell Signaling, USA), CEMIP (1:5000, 45750002, Novus Biological, USA), β-catenin (1:1000, #8480, Cell Signaling, USA), TCF3 (1:1000, #12258, Cell Signaling, USA) and WDR5 (1:1000, #13105, Cell Signaling, USA).

Techniques: Expressing, RNA Sequencing, Gene Expression, Comparison, Quantitative RT-PCR, Western Blot, Immunohistochemistry

Journal: Experimental & Molecular Medicine

Article Title: Decreased expression of ATF3, orchestrated by β-catenin/TCF3, miR-17-5p and HOXA11-AS, promoted gastric cancer progression via increased β-catenin and CEMIP

doi: 10.1038/s12276-021-00694-9

Figure Lengend Snippet: a , b shATF3 lentivirus or control lentivirus was individually transduced into AGS and HGC-27 cells, respectively. The knockdown efficiency was validated, and the proliferation capacities were detected by CCK-8 ( a ) and colony formation assays in soft agar ( b ), with bar charts showing the colony numbers. The results are expressed as the mean ± s.d. of 3 independent experiments. c , d ATF3 lentivirus or control was individually transduced into MGC-803 and MKN-45 cells. The overexpression efficiency was confirmed, and the proliferation capacities were detected by CCK-8 ( c ) and colony formation assays in soft agar ( d ), with bar charts showing the colony numbers. The results are expressed as the mean ± s.d. of 3 independent experiments. e , f Tumor growth curves ( e ) of ATF3-knockdown AGS and HGC-27 cells (or negative control cells) in the xenograft mouse model based on tumor size. Moreover, tumor nodules ( f - i ) were collected, and tumor weights ( f - ii ) were recorded to highlight the growth difference under the influence of ATF3 knockdown. The values shown are the mean ± s.d. of five tumors. g ATF3 affected peritoneal spreading in nude mice. Representative images of nude mice intraperitoneally injected with ATF3-knockdown AGS cells (i) or ATF3-overexpressing MGC-803 cells (ii). Control lentivirus-infected cells were used as the corresponding control. Quantification of the peritoneal nodules is shown in the bar graph (iii, iv; lower panels). Data are presented as the mean ± s.d. of five mice, Scale bar: 5 mm. h ATF3-knockdown AGS cells or control cells were injected into the tail vein of nude mice ( n = 5). Representative images of lung metastasis models eight weeks after tail vein injection are shown (i), and the number of metastatic lung nodules was counted (ii). Data are presented as the mean ± s.d. of five mice. i Histological analysis showed ATF3 expression levels in metastatic GC cells in lung tissue sections from the experimental and control groups. Scale bar: 20 μm. * p < 0.05; ** p < 0.01 by Student’s t test compared with t he control group.

Article Snippet: Western blots were performed as described with primary antibodies targeting ATF3 (1:1000, #33593, Cell Signaling, USA), CEMIP (1:5000, 45750002, Novus Biological, USA), β-catenin (1:1000, #8480, Cell Signaling, USA), TCF3 (1:1000, #12258, Cell Signaling, USA) and WDR5 (1:1000, #13105, Cell Signaling, USA).

Techniques: Control, Knockdown, CCK-8 Assay, Over Expression, Negative Control, Injection, Infection, Expressing

Journal: Experimental & Molecular Medicine

Article Title: Decreased expression of ATF3, orchestrated by β-catenin/TCF3, miR-17-5p and HOXA11-AS, promoted gastric cancer progression via increased β-catenin and CEMIP

doi: 10.1038/s12276-021-00694-9

Figure Lengend Snippet: a Mean-centered hierarchical clustering of 776 transcripts that were altered (≥ 2-fold change, p < 0.05) in shATF3 lentivirus-treated cells (shATF3) compared to control lentivirus-treated cells (shCtrl), with three repeats. b , c KEGG analysis of altered transcripts with upregulated or downregulated expression. d , e The mRNA levels of the identified genes were selectively confirmed by RT-qPCR in GC cells with ATF3 knockdown ( d ) or ATF3 overexpression (e). f Hierarchical clustering of 264 transcripts with altered expression (≥2-fold or ≤ −2-fold changes, p < 0.05) in ATF3 low GC tissues compared with paired adjacent ATF3 high normal tissues. g Correlation between the mRNA levels of ATF3 and those of β-catenin (i) or CEMIP in the GEO GSE66229 dataset. h , i Promoter activity of β-catenin ( h ) and CEMIP (i) as measured by luciferase reporter assays in HEK293 cells cotransfected with the indicated promoter regions upstream of luciferase and either ATF3 or empty vector, respectively. The data are presented as the mean ± s.d. j Western blot analysis of ATF3, β-catenin and CEMIP expression in ATF3-knockdown AGS cells and control cells. k Western blot analysis of ATF3, β-catenin and CEMIP expression in ATF3-overexpressing BGC-803 and MKN-45 cells. l Colony formation assay in ATF3-knockdown AGS and HGC-27 cells upon β-catenin inhibition. m Transwell migration and Matrigel invasion assays were performed to determine the effect of CEMIP inhibition on the migration and invasion of ATF3-knockdown AGS cells. The data are presented as the mean ± s.d., n = 3. * p < 0.05; ** p < 0.01, *** p < 0.001 and n.s. (not significant) by Student’s t test compared with the control group.

Article Snippet: Western blots were performed as described with primary antibodies targeting ATF3 (1:1000, #33593, Cell Signaling, USA), CEMIP (1:5000, 45750002, Novus Biological, USA), β-catenin (1:1000, #8480, Cell Signaling, USA), TCF3 (1:1000, #12258, Cell Signaling, USA) and WDR5 (1:1000, #13105, Cell Signaling, USA).

Techniques: Control, Expressing, Quantitative RT-PCR, Knockdown, Over Expression, Activity Assay, Luciferase, Plasmid Preparation, Western Blot, Colony Assay, Inhibition, Migration

Journal: Experimental & Molecular Medicine

Article Title: Decreased expression of ATF3, orchestrated by β-catenin/TCF3, miR-17-5p and HOXA11-AS, promoted gastric cancer progression via increased β-catenin and CEMIP

doi: 10.1038/s12276-021-00694-9

Figure Lengend Snippet: a RT-qPCR analysis of TCF3 mRNA expression in our GC sample set. b , c Correlation between the mRNA levels of β-catenin and TCF3 in the TCGA STAD specimens and the GEO GSE66229 dataset. d , e Correlations between the mRNA levels of TCF3 and ATF3 in the TCGA STAD specimens ( d ) and the GEO GSE66229 dataset (e). f Correlation between the mRNA levels of β-catenin and TCF3 in our sample set. g , h Correlations between the mRNA levels of ATF3 and β-catenin ( g ) or TCF3 (h) in our sample set. i , j qPCR and western blot analysis of TCF3 mRNA ( i ) and protein expression ( j - i , - ii ), respectively, in β-catenin-knockdown BGC-803 and MKN-45 cells. k The ChIP assay was performed to detect β-catenin occupancy in the TCF3 promoter region, and the IgG group was used as a negative control. l TCF3 promoter activity was measured by luciferase reporter assays in HEK293 cells cotransfected with β-catenin or empty vector. Data are presented as the mean ± s.d. n = 3. m , n RT-qPCR and western blot analysis of ATF3 mRNA ( m) and protein expression ( n - i , - ii ), respectively, in TCF3-knockdown MGC-803 and MKN-45 cells. o ChIP assays were performed to detect TCF3 occupancy in the ATF3 promoter region, and the IgG group was used as a negative control. p ATF3 promoter activity was measured by luciferase reporter assays in HEK293 cells cotransfected with TCF3 or empty vector. The data are presented as the mean ± s.d. q The protein expression levels of TCF3 and ATF3 were detected in consecutive paraffin sections of tissues from the GC TMA G10 database by IHC staining. r Correlation between the IHC scores of ATF3 and TCF3 in the GC TMA G10 specimens as detected by IHC staining. * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student’s t test compared with the paired normal or control group.

Article Snippet: Western blots were performed as described with primary antibodies targeting ATF3 (1:1000, #33593, Cell Signaling, USA), CEMIP (1:5000, 45750002, Novus Biological, USA), β-catenin (1:1000, #8480, Cell Signaling, USA), TCF3 (1:1000, #12258, Cell Signaling, USA) and WDR5 (1:1000, #13105, Cell Signaling, USA).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Knockdown, Negative Control, Activity Assay, Luciferase, Plasmid Preparation, Immunohistochemistry, Control

Journal: Experimental & Molecular Medicine

Article Title: Decreased expression of ATF3, orchestrated by β-catenin/TCF3, miR-17-5p and HOXA11-AS, promoted gastric cancer progression via increased β-catenin and CEMIP

doi: 10.1038/s12276-021-00694-9

Figure Lengend Snippet: a Heatmap of altered ATF3-targeted microRNAs in ATF3 low GC tissues compared with paired adjacent ATF3 high normal tissues. The red dotted frame shows specifically increased ATF3-targeted microRNAs detected in ATF3 low GC tissues. b Correlations between the expression levels of ATF3 and miR-17, miR-590 or miR-331 in TCGA STAD samples. c Correlations between the expression levels of ATF3 and miR-17-5p, miR-590-5p or miR-331-3p in our GC samples. d A luciferase reporter assay was used to determine the interaction between miRNA and the specific binding site on the ATF3 3′UTR. HEK293 cells were cotransfected with an ATF3 3′UTR construct and either miR-17-5p mimics or control mimics. Luciferase activity is shown as relative luciferase activity normalized to that of Renilla luciferase. Data are expressed as the mean ± s.d. of 3 independent transfections. e Western blot analysis of ATF3 levels in MGC-803 and MKN-45 cells infected individually with miR-17-5p-inhibitor lentivirus or control lentivirus. β-actin served as an endogenous control ( e - i ). Densitometry analysis of protein bands was performed with ImageJ, and the normalized ATF3 band intensity in control lentivirus-infected GC cells was set as 1 ( e - ii ). f Detection of miR-17-5p and ATF3 expression in consecutive paraffin sections of GC tissues from the TMA GC1-a dataset by fluorescence in situ hybridization (FISH) and IHC staining, respectively. Representative images of samples from three GC patients are shown. FISH and histological analysis showed the fluorescence of the miR-17-5p probe and ATF3 IHC staining in the same tissue site in consecutive sections of GC tissues; scale bar: 20 μm. g The expression level of ATF3 in GC tissues with high or low expression of miR-17-5p was statistically analyzed. * p < 0.05 and ** p < 0.01 by Student’s t test compared with the corresponding control group.

Article Snippet: Western blots were performed as described with primary antibodies targeting ATF3 (1:1000, #33593, Cell Signaling, USA), CEMIP (1:5000, 45750002, Novus Biological, USA), β-catenin (1:1000, #8480, Cell Signaling, USA), TCF3 (1:1000, #12258, Cell Signaling, USA) and WDR5 (1:1000, #13105, Cell Signaling, USA).

Techniques: Expressing, Luciferase, Reporter Assay, Binding Assay, Construct, Control, Activity Assay, Transfection, Western Blot, Infection, Fluorescence, In Situ Hybridization, Immunohistochemistry

Journal: Experimental & Molecular Medicine

Article Title: Decreased expression of ATF3, orchestrated by β-catenin/TCF3, miR-17-5p and HOXA11-AS, promoted gastric cancer progression via increased β-catenin and CEMIP

doi: 10.1038/s12276-021-00694-9

Figure Lengend Snippet: a Heatmap of altered lncRNAs (fold change > 4, p < 0.05) in ATF3 low GC tissues compared with paired adjacent normal tissues. Red arrows indicate a significant increase in lncRNA HOXA11-AS expression in ATF3 low GC tissues. b The expression level of HOXA11-AS in GC tissues compared with that in paired normal gastric tissues as detected by RT-qPCR in our GC samples. c Correlation between the mRNA levels of HOXA11-AS and ATF3 in the GEO GSE35809 dataset (i) and in our GC samples (ii). d The expression level of HOXA11-AS was detected by RT-qPCR in HOXA11-AS-knockdown MGC-803 and MKN-45 cells. e ATF3 mRNA (i) and protein levels (ii) were detected by RT-qPCR and western blot, respectively, in HOXA11-AS-knockdown MGC-803 and MKN-45 cells, with β-actin serving as an endogenous control. f RT-qPCR and western blotting were performed to detect the mRNA (i) and protein (ii) levels, respectively, of β-catenin in HOXA11-AS-knockdown MGC-803 and MKN-45 cells, with β-actin serving as an endogenous control. g RIP analyses of HOXA11-AS1 and its predicted binding partners in MGC-803 (i) and MKN-45 cells (ii). h The colocalization of HOXA11-AS and TCF3 was analyzed by concomitant FISH and IF in MGC-803 and MKN-45 cells. Red, HOXA11-AS; Green, TCF3; blue, DAPI. Scale bar: 10 μm. i Western blotting analyses of TCF3 expression in MKN-45 cells subjected to pull-down with full-length HOXA11-AS. j ChIP-qPCR analysis of TCF3, EZH2, and H3K27me3 occupancy in the ATF3 promoter in MGC-803 and MKN-45 cells. k ChIP-qPCR analysis of TCF3, EZH2, and H3K27me3 occupancy in the ATF3 promoter in HOXA11-AS-knockdown MGC-803 cells. * p < 0.05; ** p < 0.01 and *** p < 0.001 by Student’s t test compared with the corresponding control group.

Article Snippet: Western blots were performed as described with primary antibodies targeting ATF3 (1:1000, #33593, Cell Signaling, USA), CEMIP (1:5000, 45750002, Novus Biological, USA), β-catenin (1:1000, #8480, Cell Signaling, USA), TCF3 (1:1000, #12258, Cell Signaling, USA) and WDR5 (1:1000, #13105, Cell Signaling, USA).

Techniques: Expressing, Quantitative RT-PCR, Knockdown, Western Blot, Control, Binding Assay, ChIP-qPCR

Journal: Experimental & Molecular Medicine

Article Title: Decreased expression of ATF3, orchestrated by β-catenin/TCF3, miR-17-5p and HOXA11-AS, promoted gastric cancer progression via increased β-catenin and CEMIP

doi: 10.1038/s12276-021-00694-9

Figure Lengend Snippet: a Correlation between the mRNA levels of TCF3 and WDR5 in the TCGA STAD specimens (i) and the GEO GSE66229 dataset (ii). b ChIP-qPCR analysis of TCF3 occupancy in the WDR5 promoter in MGC-803 and MKN-45 cells. c The promoter activity of WDR5 was measured by luciferase reporter gene analysis in HEK293T cells cotransfected with vector containing the target promoter upstream of luciferase and TCF3 or empty vector control. The data are presented as the mean ± s.d. d , e RT-qPCR and western blot analysis of WDR5 mRNA ( d ) and protein levels ( e - i , - ii ), respectively, in TCF3 knockdown MGC-803 and MKN-45 cells. f Correlations between the expression level of WDR5 and HOXA11-AS (i), miR-17 (ii), or ATF3 (iii) in the TCGA STAD specimens. g , h ChIP-qPCR analysis of WDR5 and H3K4me3 occupancy in the HOXA11-AS promoter ( g ) and miR-17 promoter ( h ) in MGC-803 and MKN-45 cells. i Promoter activity of β-catenin as measured by luciferase reporter assay in HEK293T cells cotransfected with vector containing the β-catenin promoter upstream of the luciferase gene and either WDR5 or empty vector controls. The data are presented as the mean ± s.d. n = 3. j , k RT-qPCR analysis of HOXA11-AS ( j ) and miR-17-5p RNA levels ( k ) in WDR5-knockdown MGC-803 (i) and MKN-45 cells (ii). l A schematic diagram illustrating the proposed molecular mechanisms of ATF3 inhibition in GC cells and its biological role in the progression of GC. * p < 0.05; ** p < 0.01 and *** p < 0.001 by Student’s t test compared with the control group.

Article Snippet: Western blots were performed as described with primary antibodies targeting ATF3 (1:1000, #33593, Cell Signaling, USA), CEMIP (1:5000, 45750002, Novus Biological, USA), β-catenin (1:1000, #8480, Cell Signaling, USA), TCF3 (1:1000, #12258, Cell Signaling, USA) and WDR5 (1:1000, #13105, Cell Signaling, USA).

Techniques: ChIP-qPCR, Activity Assay, Luciferase, Plasmid Preparation, Control, Quantitative RT-PCR, Western Blot, Knockdown, Expressing, Reporter Assay, Inhibition