Journal: Molecular and Cellular Biology

Article Title: Shared Gene Targets of the ATF4 and p53 Transcriptional Networks

doi: 10.1080/10985549.2023.2229225

Figure Lengend Snippet: ATF3 is a p53 target gene that is activated via the ISR in a p53-independent manner. Western blot analysis of (A) ATF3 and (B) p53 with GAPDH as a loading control in HCT116 p53 WT (left) and p53 null cells (right) following a 6 h treatment with DMSO, 5 μM Nutlin-3A (NUT), 100 μM etoposide (ETOP), 2 μM tunicamycin (TM) or 2 mM histidinol (HisOH). Gene expression analysis of the (C) ATF3 gene (D) CDKN1A gene and (E) ASNS gene in HCT116 p53 WT (black) and HCT116 p53 null (pink) cells in response to 6 h treatment with stimuli. All statistical comparisons were computed using a one-way ANOVA test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: For cell line treatments, cells were cultured for times indicated in each experimental figure/legend with either 5 μM nutlin-3A (Millipore Sigma, #45-SML0580) to stabilize p53 activation, 100 μM etoposide (Thermo Scientific, #J63651.MC), 2 μM tunicamycin (Thermo Scientific, #J62217.MA) or 2 mM histidinol (Acros Organics, #AC228831000).

Techniques: Western Blot, Control, Gene Expression

Journal: Molecular and Cellular Biology

Article Title: Shared Gene Targets of the ATF4 and p53 Transcriptional Networks

doi: 10.1080/10985549.2023.2229225

Figure Lengend Snippet: ATF4 and p53 independently regulate expression of ATF3 . ATF4 protein (A to C) and mRNA (D to F) expression analysis by Western blot and qRT-PCR, respectively, in HCT116 WT or p53 null cells (A, D), with shRNA constructs targeting control region (control shRNA) or ATF4 (ATF4 shRNA) (B and E), or HAP1 WT or HAP1 ATF4KO cells (C and F). Gene expression analysis of ATF3 (G and H) or ASNS (I and J) in HCT116 ATF4 shRNA cells (G and I) or HAP1 ATF4KO cells (H and J). Cells were harvested 6 h post-treatment with DMSO, 10 μM nutlin-3A (NUT), 100 μM etoposide (ETOP), 2 μM tunicamycin (TM) or 2 mM histidinol (HisOH). All statistical comparisons were computed using a one-way ANOVA test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: For cell line treatments, cells were cultured for times indicated in each experimental figure/legend with either 5 μM nutlin-3A (Millipore Sigma, #45-SML0580) to stabilize p53 activation, 100 μM etoposide (Thermo Scientific, #J63651.MC), 2 μM tunicamycin (Thermo Scientific, #J62217.MA) or 2 mM histidinol (Acros Organics, #AC228831000).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, shRNA, Construct, Control, Gene Expression

Journal: Molecular and Cellular Biology

Article Title: Shared Gene Targets of the ATF4 and p53 Transcriptional Networks

doi: 10.1080/10985549.2023.2229225

Figure Lengend Snippet: ATF4 and p53 occupy distinct regulatory regions in the ATF3 gene locus. (A) Known motif enrichment analysis of the high-confidence peak set reveals the predicted ATF4 motif as the most highly enriched motif within this dataset. (B) De novo motif analysis of high-confidence peak set shows enrichment of ATF4 motif. (C) Enrichment of CUT&RUN sequencing tags for the 7723 high-confidence ATF4 peaks after 6 h drug treatments as indicated from −1 kb and +1 kb from peak center. (D) Genome browser view of the ATF3 gene locus displaying ATF4 CUT&RUN data (black) and p53 ChIP-Seq data (green) (scaled to 1 as the maximum value for ATF4 or p53) following 6 h treatment with various stress stimuli: DMSO (vehicle control), 5 μM nutlin-3A (NUTLIN), 100 μM etoposide (ETOP), 2 μM tunicamycin (TM), or 2 mM histidinol (HisOH). (E to G) The top five most enriched results from chiprenrich for ATF4 CUT&RUN high-confidence peaks (from this manuscript) or nutlin-induced p53 ChIP-seq peaks (from Ref. ) for (E) the mSigDB v6.0 Hallmark , (F) KEGG v. 3.2.3 , or (G) REACTOME v. 61 gene sets. P values are log 10 (1/Bonferroni-corrected P value). Full data tables for enrichment results can be found in Table S5 .

Article Snippet: For cell line treatments, cells were cultured for times indicated in each experimental figure/legend with either 5 μM nutlin-3A (Millipore Sigma, #45-SML0580) to stabilize p53 activation, 100 μM etoposide (Thermo Scientific, #J63651.MC), 2 μM tunicamycin (Thermo Scientific, #J62217.MA) or 2 mM histidinol (Acros Organics, #AC228831000).

Techniques: Sequencing, ChIP-sequencing, Control

Journal: Molecular and Cellular Biology

Article Title: Shared Gene Targets of the ATF4 and p53 Transcriptional Networks

doi: 10.1080/10985549.2023.2229225

Figure Lengend Snippet: ATF3 induction by the ISR does not require the upstream enhancer element bound by p53. (A) Normalized luciferase values driven by the upstream ATF3 DNase hypersensitivity sites (DHS): ATF4-bound DHS, p53-bound DHS, p53RE mutant, and the minimal promoter (negative control), in response to 16 h treatment with DMSO, 5 μM nutlin-3A (NUT) or 2 μM tunicamycin (Tm) in HCT116 p53 WT and p53 null cells. (B) Normalized luciferase values driven by the (−104/+36) ATF3 promoter sequence (WT ATF3 promoter) and constructs containing mutations in specific ATF4 response elements: CARE, CRE, CARE/CRE, in response to 16 h treatment with DMSO, 2 μM tunicamycin (Tm), or 2 mM histidinol (HisOH) in HAP1 parental and ATF4KO cells. Luciferase reporters with relevant motif positions and sequences are illustrated above the corresponding bar chart (A and B), genomic locations of the ATF3 promoter and DHS are reported in Table S1 . (C) Genome browser view of the ATF3 gene locus displaying the location of dCas9-KRAB gRNA targets and the genomic coordinates spanning these targets relevant to panel D. (D) RT-qPCR analysis of the ATF3 gene in response to 6 h treatment with DMSO, 100 μM etoposide (ETOP) or 2 μM tunicamycin (TM) in HCT116 p53 WT cells where dCas9-KRAB is targeting regions at off-target sites at a control FGF2 enhancer (blue) or intergenic region (orange and green), the p53-bound ATF3 enhancer element (purple) or ATF3 promoter (red) for transcriptional repression. RT-qPCR analysis of the (E) ASNS gene, and (F) CDKN1A / p21 gene, following a 6 h treatment with various stress stimuli. Statistical comparisons for nascent expression levels were computed using a one-way ANOVA test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: For cell line treatments, cells were cultured for times indicated in each experimental figure/legend with either 5 μM nutlin-3A (Millipore Sigma, #45-SML0580) to stabilize p53 activation, 100 μM etoposide (Thermo Scientific, #J63651.MC), 2 μM tunicamycin (Thermo Scientific, #J62217.MA) or 2 mM histidinol (Acros Organics, #AC228831000).

Techniques: Luciferase, Mutagenesis, Negative Control, Sequencing, Construct, Quantitative RT-PCR, Control, Expressing

Journal: Molecular and Cellular Biology

Article Title: Shared Gene Targets of the ATF4 and p53 Transcriptional Networks

doi: 10.1080/10985549.2023.2229225

Figure Lengend Snippet: Global transcriptome analysis identifies common gene regulatory targets of the p53 GRN and the ISR. (A) Intersection of genes upregulated (any fold-change relative to DMSO, Bonferroni-adjusted P value <0.05) in HCT116 p53 WT cells treated with 5 μM nutlin-3A, 100 μM etoposide, 2 μM tunicamycin, and 2 μM histidinol, when compared to vehicle control (DMSO) for 6 h. (B) Bar plots representing a fraction of genes identified from the RNA-seq experiment as upregulated (yellow), downregulated (blue), or not regulated (white) in response to various stimuli that have a CUT&RUN-defined ATF4 binding event within a binned distance indicated on the x -axis. (C) Causal inference engine predictions of the top five putative upstream regulators of genes from the TRRUST database induced by tunicamycin, histidinol, or etoposide treatment (as determined using the Fisher’s exact test for significance). , Complete causal inference engine results for the STRING database for both WT and HCT116 p53 null cells can be found in Fig. S3 and Table S4 . (D) Gene ontology analysis of the genes commonly upregulated and downregulated (any fold-change relative to DMSO, Bonferroni-adjusted P value <0.05), in response to various stress stimuli. (E) Heatmap and hierarchical clustering results (one minus Pearson, average linkage) displaying fold change values for the 26 common targets identified in panel A. (F) Table displaying the gene symbols for the 26 common target Ensembl gene IDs identified in panel A and matching row names in panel E. Genes validated in by RT-qPCR are depicted in bold font.

Article Snippet: For cell line treatments, cells were cultured for times indicated in each experimental figure/legend with either 5 μM nutlin-3A (Millipore Sigma, #45-SML0580) to stabilize p53 activation, 100 μM etoposide (Thermo Scientific, #J63651.MC), 2 μM tunicamycin (Thermo Scientific, #J62217.MA) or 2 mM histidinol (Acros Organics, #AC228831000).

Techniques: Control, RNA Sequencing, Binding Assay, Quantitative RT-PCR

Journal: Molecular and Cellular Biology

Article Title: Shared Gene Targets of the ATF4 and p53 Transcriptional Networks

doi: 10.1080/10985549.2023.2229225

Figure Lengend Snippet: Parallel stress-dependent networks converge at activation of a common set of target genes. RT-qPCR analysis of the ATF3, GADD45A , SESN2, and GDF15 gene in (A to D) HCT116 p53 WT and p53 null cells, (E to H) MCF10A p53 WT and p53 null cells, and (I to L) HAP1 parental and ATF4KO cells, following a 6 h treatment with DMSO, 100 μM etoposide (ETOP), or 2 μM tunicamycin (Tm). All statistical comparisons were computed using a one-way ANOVA test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: For cell line treatments, cells were cultured for times indicated in each experimental figure/legend with either 5 μM nutlin-3A (Millipore Sigma, #45-SML0580) to stabilize p53 activation, 100 μM etoposide (Thermo Scientific, #J63651.MC), 2 μM tunicamycin (Thermo Scientific, #J62217.MA) or 2 mM histidinol (Acros Organics, #AC228831000).

Techniques: Activation Assay, Quantitative RT-PCR

Journal: Molecular and Cellular Biology

Article Title: Shared Gene Targets of the ATF4 and p53 Transcriptional Networks

doi: 10.1080/10985549.2023.2229225

Figure Lengend Snippet: GADD45A as a reporter system to study DDR- and ISR-dependent enhancers. (A) Genome browser view with GADD45A locus displaying ATF4 CUT&RUN and p53 ChIP-Seq data in HCT116 WT cell line following 6 h treatment with DMSO, 2 mM histidinol (HisOH), 2 μM tunicamycin (TM), and 5 µM nutlin-3A (NUT). Putative p53RE, ATF4 motif and GADD45A intron 3 enhancer locations are indicated on the bottom, DNaseI hypersensitive sites (DHS) are marked in gray/black. (B) Schematic representation of the GADD45A-nLuc reporter construct with relevant enhancer sequence motifs highlighted in panel C. (D) Normalized luciferase expression values using GADD45A-nLuc reporter transfected into HCT116 WT cell line and 16 h treatment with DMSO, nutlin-3A and tunicamycin as indicated in the legend. Reporter constructs included wild-type, a negative control with 250 bp enhancer deletion (“No Enhancer”) and various ATF4, AP1 and p53RE motif mutations alone or in combination as indicated in the table below (“WT” or “mutant” in gray). Specific mutations in motifs are indicated in panel C. Statistical comparisons were generated using one-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: For cell line treatments, cells were cultured for times indicated in each experimental figure/legend with either 5 μM nutlin-3A (Millipore Sigma, #45-SML0580) to stabilize p53 activation, 100 μM etoposide (Thermo Scientific, #J63651.MC), 2 μM tunicamycin (Thermo Scientific, #J62217.MA) or 2 mM histidinol (Acros Organics, #AC228831000).

Techniques: ChIP-sequencing, Construct, Sequencing, Luciferase, Expressing, Transfection, Negative Control, Mutagenesis, Generated

Journal: Molecular and Cellular Biology

Article Title: Shared Gene Targets of the ATF4 and p53 Transcriptional Networks

doi: 10.1080/10985549.2023.2229225

Figure Lengend Snippet: Other STARR-seq-identified motifs contribute only to basal GADD45A enhancer activity. (A) Schematic representation of the GADD45A-nLuc reporter construct with relevant enhancer sequence motifs highlighted in panel B. (C) Normalized luciferase expression values using GADD45A-nLuc reporter transfected into HCT116 WT cell line and 16 h treatment with DMSO, nutlin-3A and tunicamycin as indicated in the legend. Reporter constructs included wild-type (WT) construct, 250 bp enhancer deletion (“No Enhancer”) as a negative control and various predicted transcription factor motif mutations as indicated on the x -axis. Specific mutations in transcription factor motifs based on the STARR-seq screen are indicated in panel B. Statistical comparisons were generated using one-way ANOVA: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: For cell line treatments, cells were cultured for times indicated in each experimental figure/legend with either 5 μM nutlin-3A (Millipore Sigma, #45-SML0580) to stabilize p53 activation, 100 μM etoposide (Thermo Scientific, #J63651.MC), 2 μM tunicamycin (Thermo Scientific, #J62217.MA) or 2 mM histidinol (Acros Organics, #AC228831000).

Techniques: Activity Assay, Construct, Sequencing, Luciferase, Expressing, Transfection, Negative Control, Generated

Journal: Oncotarget

Article Title: Disulfide bond disrupting agents activate the unfolded protein response in EGFR- and HER2-positive breast tumor cells

doi: 10.18632/oncotarget.15952

Figure Lengend Snippet: (A) The specified cell lines were treated with the indicated RBF3 concentrations for 24 hours and analyzed for the ER stress marker GRP78 by immunoblot (left panel). The right panel indicates the status of EGFR or HER2 expression and DDA sensitivity. (B) Proliferation of MDA-MB-468 cells treated for 24 hours with the indicated concentrations of RBF3 in the presence or absence of 20 ng/ml EGF as assessed by tritiated thymidine incorporation. Results are presented as the mean ± standard deviation of triplicate determinations. (C) DDA sensitive or resistant cell lines were treated for 24 hours with increasing concentrations of RBF3 and extracts were analyzed by immunoblot for markers related to ER stress. (D) The time course of RBF3 responses in MDA-MB-468 cells was compared with that of the ER stress inducers tunicamycin (500 ng/ml) and thapsigargin (400 nM) by immunoblot analysis. (E) Luciferase reporter assays measuring the impact of ectopically expressed ATF6 and 20 μM RBF3 on the activity of an ATF6-responsive promoter construct. Results are normalized to micrograms of protein extract assayed, and are presented as the mean ± standard deviation of triplicate determinations. (F) Extracts from HEK 293 cells transiently transfected as indicated and treated with or without 20 μM RBF3 for 24 hours were analyzed by immunoblot.

Article Snippet: The following reagents were purchased from the indicated sources: tunicamycin, 2-deoxyglucose: Sigma-Aldrich (St. Louis, MO); 2-aminoethoxydiphenyl borate (2-APB): StressMarq Biosciences (Cadboro Bay, Victoria, Canada); thapsigargin: AdipoGen (San Diego, CA); Puromycin, Rapamycin, Cycloheximide: EMD Biosciences (Darmstadt, Germany); Gefitinib, Lapatinib, SAHA: Selleck Chemicals (Houston, TX); dithiothreitol: Fisher Scientific (Pittsburgh, PA).

Techniques: Marker, Western Blot, Expressing, Standard Deviation, Luciferase, Activity Assay, Construct, Transfection

Journal: Oncotarget

Article Title: Disulfide bond disrupting agents activate the unfolded protein response in EGFR- and HER2-positive breast tumor cells

doi: 10.18632/oncotarget.15952

Figure Lengend Snippet: (A) MDA-MB-468 and BT474 cells were treated for 24 hours with 20 μM RBF3, 100 μM 2-Aminoethoxydiphenyl Borate (2-APB), or 4 mM 2-deoxyglucose (2-DOG) in the indicated combinations and cell extracts were analyzed by immunoblot. (B) MDA-MB-468 cells were treated for 24 hours with 20 μM RBF3, 400 nM thapsigargin, 500 ng/ml tunicamycin or dithiothreitol (DTT) at the indicated concentrations and cell extracts were analyzed by immunoblot. The effects of these same treatments on the splicing of the mRNA coding for XBP1s was assessed by reverse transcription of mRNA followed by DNA amplification (RT-PCR). (C) BT474 cells were treated for 24 hours with 20 μM RBF3, 400 nM thapsigargin, 500 ng/ml tunicamycin, or DTT at the indicated concentrations, and cell extracts were analyzed by immunoblot. The effects of these same treatments on the splicing of the mRNA coding for XBP1s was assessed by reverse transcription of mRNA followed by DNA amplification (RT-PCR). (D) Wild type or eIF2α[S51A] double knock-in mutant MEFs were treated for 24 hours with 20 μM RBF3, 400 nM thapsigargin, 500 ng/ml tunicamycin, 5 mM DTT, or vehicle and cell extracts were prepared and analyzed by immunoblot.

Article Snippet: The following reagents were purchased from the indicated sources: tunicamycin, 2-deoxyglucose: Sigma-Aldrich (St. Louis, MO); 2-aminoethoxydiphenyl borate (2-APB): StressMarq Biosciences (Cadboro Bay, Victoria, Canada); thapsigargin: AdipoGen (San Diego, CA); Puromycin, Rapamycin, Cycloheximide: EMD Biosciences (Darmstadt, Germany); Gefitinib, Lapatinib, SAHA: Selleck Chemicals (Houston, TX); dithiothreitol: Fisher Scientific (Pittsburgh, PA).

Techniques: Western Blot, Reverse Transcription, DNA Amplification, Reverse Transcription Polymerase Chain Reaction, Knock-In, Mutagenesis

Journal: JCI insight

Article Title: ClC-Kb pore mutation disrupts glycosylation and triggers distal tubular remodeling.

doi: 10.1172/jci.insight.175998

Figure Lengend Snippet: Figure 4. Asn-364 glycosylation is critical for ClC-Kb function. (A–C) Voltage-clamp experiment shows a reduced conductance in the presence of N-linked glycosylation inhibitor, tunicamycin, compared with vehicle-treated CHO cells (control). Data are mean ± SEM of n = 7–26 samples per condition. *P < 0.05 vs. control by unpaired Student’s t test. (D) Quantification of electrophysiological recordings from CHO cells expressing wild-type (WT) or potential glycosyla- tion-site mutants of ClC-Kb (N364L, N373L, N364L/N373L). Data are mean ± SEM of n = 6–14 samples per condition. *P < 0.05 vs. WT by 1-way ANOVA. (E) Western blot of transfected WT and glycosylation mutants of ClC-Kb in HEK293T cells (probed with anti-HA and anti-cadherin antibodies). (F) Quantitative analysis of protein expression (glycosylated and nonglycosylated) of WT vs. potential glycosylation-site mutants (blue, glycosylated; black, nonglycosylated). Data are mean ± SEM of n = 3 independent experiments. *P < 0.05 vs. glycosylated band for WT; #P < 0.05 for nonglycosylat- ed band vs. WT by 1-way ANOVA. M, marker.

Article Snippet: We obtained current-voltage (I-V) relationships by monitoring channel activity at applied holding potential from –80 mV to +70 mV, with a step of 10 mV/s in the presence or absence of 5 μg/mL tunicamycin (Tocris Bioscience), an inhibitor of N-linked glycosylation, as indicated.

Techniques: Glycoproteomics, Control, Expressing, Western Blot, Transfection, Marker

Journal: iScience

Article Title: MAVS phosphorylation acts as a cellular stress sensor that modulates antiviral immunity

doi: 10.1016/j.isci.2025.113256

Figure Lengend Snippet: ER stress increases expression of type I IFNs via phosphorylation of MAVS (A and B) HEK293T cells transiently transfected for 20 h with expression vectors for Myc-tagged WT mMAVS were treated with 100 μg/mL of tunicamycin (Tn) for 3 h. The lysates were then subjected to immunoprecipitation (IP) with and anti-Myc antibody. The resulting precipitates were subjected to immunoblot analysis, together with the original cell lysates (Total), with antibodies specific for phospho-S 220 MAVS, Myc, p-p38, or p38 (A). Results are representative of three independent experiments. Densitometry analysis of the phospho-S 220 MAVS band relative to the Myc band in the immunoprecipitate (B). Data are expressed as the mean ± SEM from three independent experiments (∗ p < 0.05, one-sample t - test). (C and D) HEK293T cells transiently transfected for 20 h with expression vectors for Myc-tagged WT mMAVS were treated with 3 μM of thapsigargin (Tg) for 3h. The lysates were then subjected to IP with an ant-Myc antibody. The resulting precipitates were then subjected to immunoblot analysis, together with the original cell lysates (Total), with antibodies specific for phospho-S 220 MAVS, Myc, p-p38, or p38 (C). Results are representative of three independent experiments. Densitometry analysis of the phospho-S 220 MAVS band relative to the Myc band in the immunoprecipitate (D). Data are expressed as the mean ± SEM from three independent experiments (∗ p < 0.05, one-sample t - test). (E and F) MAVS KO MEFs were reconstituted with MAVS WT or the 2SA mutant and treated with 100 μg/mL of Tn for 3h. Cells were then transfected for 3 h with 0.25 μg/mL poly(I:C) and subjected to reverse transcription and quantitative polymerase chain reaction (RT-qPCR) analysis to detect Ifna (E) or Ifnb1 (F) mRNA. Data are expressed as the mean ± SEM of four independent experiments (∗ p < 0.05, ∗∗ p < 0.01, non-significant (NS), one-way ANOVA with Tukey’s multiple comparisons test). (G and H) MAVS KO MEFs were reconstituted with MAVS WT or the 2SA mutants and treated for 3 h with 3 μM of Tg. Cells were then transfected with 0.25 μg/mL poly(I:C) for 3 h, and subjected to reverse transcription and quantitative polymerase chain reaction (RT-qPCR) analysis to detect Ifna (G) or Ifnb1 (H) mRNA. Data are expressed as the mean ± SEM of six independent experiments (∗ p < 0.05, ∗∗ p < 0.01, non-significant (NS), one-way ANOVA with Tukey’s multiple comparisons test). (I and J) MAVS KO MEFs were reconstituted with MAVS WT or the 2SA mutant treated for 3 h with 100 μg/mL of Tn (I) or 3 μM of Tg (J). cells were then infected with VSV for 3 h, and subjected to reverse transcription and quantitative polymerase chain reaction (RT-qPCR) analysis to detect Ifnb1 mRNA. Data are expressed as the mean ± SEM of five independent experiments (∗ p < 0.05, non-significant (NS), one-way ANOVA with Tukey’s multiple comparisons test).

Article Snippet: Tunicamycin , LKT Labs, Inc. , T8153.

Techniques: Expressing, Phospho-proteomics, Transfection, Immunoprecipitation, Western Blot, Mutagenesis, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Infection

Journal: Cell reports

Article Title: Nuclear translocation of an aminoacyl-tRNA synthetase may mediate a chronic “integrated stress response”

doi: 10.1016/j.celrep.2023.112632

Figure Lengend Snippet:

Article Snippet: Hydrogen peroxide (Millipore Sigma) was applied at 200 μM, and tunicamycin (Cayman Chemical) was applied at 5 μg/mL.

Techniques: Recombinant, Extraction, Silver Staining, CCK-8 Assay, Software