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: bioRxiv

Article Title: Endocytic control of cell-autonomous and non-cell-autonomous functions of p53

doi: 10.1101/2025.08.16.670648

Figure Lengend Snippet: A. Ctrl-KO and SNX9-KO MCF10A cells were treated with etoposide at 25 and 50 μM (indicated by triangles) and analyzed by IB for total and phosphorylated (p53 ser15 ) p53, and the p53 target genes MDM2 and CDKN1A. Vinculin (VCL), loading control. B . RT-qPCR analysis of MDM2 and CDKN1A expression in the samples described in “A”. Data are from three independent experiments and expressed as mean ± SD. *, p<0.05; **, p<0.01. C. 3D reconstruction of MCF10A-p53-KO recipient cells treated for 8 h with EVs purified from the conditioned medium of HEK-293 cells transfected with p53-HA. Recipient cells were treated with etoposide (50 μM for 8 h) and analyzed by IF using an anti-p53 antibody (green) and DAPI (blue). Bar, 20 μm. D. MCF10A-p53-KO recipient cells were treated for 8 h with etoposide (50 μM) and EVs purified from HEK-293 WT (EV) or HEK-293 p53-HA (EV p53-HA) cells as indicated. After treatment cells were harvested and analyzed by RT-qPCR for CDKN1A levels. Data are from three independent experiments and expressed as mean ± SD. * p<0.05, ** p<0.01 and *** p<0.001 vs . same condition in cells not treated with EVs (only the most relevant statistical comparisons are shown). E. Scheme of the co-culture experiment shown in in panel F. a) MCF10A-p53-KO-H2B-Cherry cells were plated onto coverslips. b) After 24 h, coverslips were harvested and seeded (c) in plates in which the indicated cell lines had been previously seeded. Cells were then treated with etoposide (50 μM) or mock-treatment for 8 h. d) Coverslips were harvested and analyzed for purity of H2B-Cherry labeled cells (Fig. S10D) and for the levels of CDKN1A mRNA (panel F). Details are in Materials and Methods. F . RT-qPCR analysis of CDKN1A levels in harvested H2B-Cherry labeled MCF10A-p53-KO cells, co-cultured as described in panel “ E ”. Data are from three independent experiments and expressed as mean ± SD. **, p<0.01; n.s., not significant (only the most relevant statistical comparisons are shown). G . SAOS2 cells were treated with EVs purified from the indicated MCF10A cell lines (see experimental scheme in Fig. S10F), and cell viability/growth was assessed indirectly by quantifying intracellular ATP levels using a luminescence-based assay. Data are from ten samples/condition from two independent experiments and expressed as mean ± SD. One-way ANOVA test: *, and ***, p < 0.05 and < 0.001, respectively, vs . SAOS2 treated with EVs derived from MCF10A-p53-KO cells.

Article Snippet: Chemicals were: FLAG peptide, cat. F3290 (Merck Life Science); HA peptide, cat. 11666975001 (Merck Life Science); NUMB peptide corresponding to amino acids 537-551 of hNUMB (Genscript); Etoposide, cat. E1383 (Merck Life Science); Trehalose Dihydrate, cat. T9531 (Merck Life Science); Chloroquine, cat. C6628 (Merck Life Science); Ionomycin, cat. I0634 (Merck Life Science); Proteinase K, P4850 (Merck Life Science); MG132, cat. 474790 (Merck Life Science); U73122, cat. 6756 (Merck Life Science); GW4869, cat. S7609 (Selleck Chemicals); brain PI(4,5)P2, cat. 840046P; brain PI(4)P, cat. 840045P; brain PS, cat. 840032C; brain PC, cat. 840053C (all from Merck Life Science).

Techniques: Control, Quantitative RT-PCR, Expressing, Purification, Transfection, Co-Culture Assay, Labeling, Cell Culture, Luminescence Assay, Derivative Assay

Journal: Nutrients

Article Title: Inhibitory Effects of Culinary Herbs and Spices on the Growth of HCA-7 Colorectal Cancer Cells and Their COX-2 Expression

doi: 10.3390/nu9101051

Figure Lengend Snippet: BLE and TE effect on proteins markers for apoptosis in HCA-7 cell line. ( a ) Western blot” Cells were treated for 24 h with bay leaf (BLE 15 μg GAE/mL), turmeric (TE 10 μg GAE/mL), and Etoposide 25 μM, which was used as a positive control for caspase-3 activation. ( a ) Quantitative analysis of Western blot bands. Protein expression was normalised against β-Actin and expressed relative to untreated control, where control is 100%; ( b ) Untreated control contained just DMEM with 10% FBS (vehicle control–ethanol was 0.4% ( v / v ), the highest amount found in the extracts). Bay leaf in ethanol (BLE), turmeric in ethanol (TE), n = 3, ±SEM.

Article Snippet: A caspase-3/7 inhibitor and Etoposide (Tocris Bioscience, Bristol, UK), a caspase activator, were used as a positive control for caspase 3 activation and a negative control, respectively.

Techniques: Western Blot, Positive Control, Activation Assay, Expressing, Control

Journal: PLOS Biology

Article Title: Interferon regulatory factor 4 mediates nonenzymatic IRE1 dependency in multiple myeloma cells

doi: 10.1371/journal.pbio.3003096

Figure Lengend Snippet: (A) Effect of IRF4, IRE1, or XBP1 silencing on in vitro spheroid growth of AMO1. Cells were stably transfected with plasmids encoding Dox-inducible shRNAs against either IRF4 (purple) or non-targeting control (blue). Growth of these cells in the absence (closed symbols) or presence (open symbols) of Dox (0.2 μg/mL) was compared to that of cells expressing shRNAs against IRE1 or XBP1. Spheroid growth, depicted as FC confluence, was monitored by time-lapse microscopy in an IncuCyte instrument and values represent mean ± SEM. (B) Effect of IRF4, IRE1, or XBP1 silencing on number of cell divisions. AMO1 shIRE1 Cl.1, shIRF4 Cl.1, or shXBP1 Cl.1 cells were stained with CFSE-type dye and incubated in the absence (filled curves) or presence (open curves) of Dox (0.2 μg/mL) and analyzed by flow cytometry. Etoposide (Eto, 25 μM, dashed line) was used as a non-proliferative control. Representative experiment out of 3 independent replicates. (C) Effect of IRF4, IRE1, or XBP1 silencing on DNA replication. AMO1 shIRE1 Cl.1, shIRF4 Cl.1, or shXBP1 Cl.1 cells were pulsed with BrdU (10 μM) and incubated in the absence (filled bars) or presence (open bars) of Dox (0.2 μg/mL) and analyzed by flow cytometry. Etoposide (Eto, 25 μM, dotted line) was used as a non-proliferative control. Data represented as mean ±SEM. (D) Effect of IRE1 or IRF4 silencing on cell cycle progression. AMO1 shIRE1 Cl.1 or shIRF4 Cl.1 cells were incubated in the absence (filled symbols) or presence (open symbols) of Dox (0.2 μg/mL) for the indicated timepoints, EtOH-fixated and PI stained before analyzed by flow cytometry. The indicated cell cycle phases were determined according to univariate (DNA content) modeling. Representative experiment out of at least 3 independent replicates. (E) Effect of IRE1 or IRF4 silencing on the rate of G2/M progression. AMO1 shIRE1 Cl.1 or shIRF4 Cl.1 cells were pre-incubated with 9 μM RO-3306 CDK1 inhibitor (synchronization to G2/M phase) in the absence or presence of Dox (0.2 μg/mL). Cells in G2/M phase were collected and their cell cycle progression during indicated time points post-sorting was analyzed by flow cytometry as before. The indicated cell cycle phases were determined according to DNA content and EdU incorporation to accurately decipher S phase. (F) Effect of IRE1 or IRF4 silencing on CDK2 activation. AMO1 shIRE1 Cl.1 or shIRF4 Cl.1 cells were incubated in the absence or presence of Dox (0.2 μg/mL) for 24 h. CDK2 was purified by immunoprecipitation. The top band is inactive CDK2 and the bottom band is the active form . Additionally, binding of the CDK2 substrate, Rb, is reduced by IRE1 or IRF4 silencing while binding of p21, the CDK inhibitor, is increased. Ig represents an isotype control for Ig detection. (G) Effect of IRE1 or IRF4 silencing on subcellular abundance of CDK2. Samples from and samples from AMO1 shIRF4 Cl.1 cells were analyzed by IB for CDK2 protein. Subcellular fractions: C—cytoplasmic, M—Membrane, SN—Soluble Nuclear, CN—Chromatin-bound Nuclear. Nuclear fractions were analyzed by IB for IRE1 and IRF4 while Cofilin, Histone H3, and Lamin B2 served as fractionation internal controls. The blots for IRE1, IRF4, Cofilin, and Lamin B2 from are shown here again for direct comparison. Data underlying this figure can be found in and .

Article Snippet: Etoposide (25 μM, #1226) was from R&D systems.

Techniques: In Vitro, Stable Transfection, Transfection, Control, Expressing, Time-lapse Microscopy, Staining, Incubation, Flow Cytometry, Activation Assay, Purification, Immunoprecipitation, Binding Assay, Membrane, Fractionation, Comparison

Journal: Toxicological sciences : an official journal of the Society of Toxicology

Article Title: Chemotherapeutic Drugs Alter Functional Properties and Proteome of Mouse Testicular Germ Cells In Vitro.

doi: 10.1093/toxsci/kfy098

Figure Lengend Snippet: Figure 2. In vitro proliferation of testicular germ cells after drugs’ exposure. (A) Fluorescent microscopic image of in vitro proliferated testicular germ cell of GFP mouse treated with etoposide, cisplatin, bleomycin,

Article Snippet: Chemotherapeutic agents or drugs named as bleomycin sulfate, etoposide and cisplatin were collected from LKT Laboratories (St Paul, Minnesota).

Techniques: In Vitro

Journal: Toxicological sciences : an official journal of the Society of Toxicology

Article Title: Chemotherapeutic Drugs Alter Functional Properties and Proteome of Mouse Testicular Germ Cells In Vitro.

doi: 10.1093/toxsci/kfy098

Figure Lengend Snippet: Figure 3. Effects of chemotherapeutic drugs on the apoptotic rate of testicular germ cells. (A) Flow cytometric determination of the apoptotic rate of drugs treated cultured germ cells after stained with Annexin V/PI. Bar graphs (B), (C), (D), and (E) represent the percentages of apoptotic germ cells due to administration of etoposide, cisplatin, bleomycin, and BEP respectively. Values are represented mean ± SEM of 6 independent experiments. Different letters (a and b) indicate significant difference (p < 0.05) compared

Article Snippet: Chemotherapeutic agents or drugs named as bleomycin sulfate, etoposide and cisplatin were collected from LKT Laboratories (St Paul, Minnesota).

Techniques: Cell Culture, Staining

Journal: Toxicological sciences : an official journal of the Society of Toxicology

Article Title: Chemotherapeutic Drugs Alter Functional Properties and Proteome of Mouse Testicular Germ Cells In Vitro.

doi: 10.1093/toxsci/kfy098

Figure Lengend Snippet: Figure 5. Effects of chemotherapeutic agents on the stemness properties of spermatogonial stem cells (SSCs). Stemness properties of SSCs were evaluated by counting donor germ cell derived colonies from recipient testes. Cultured germ cells treated with 0.05 µM etoposide, 1 µM cisplatin, 10 µM bleomycin, and 0.1 µM BEP were transplanted and colonies were counted after 2 months 5of transplantation. (A) Testes of

Article Snippet: Chemotherapeutic agents or drugs named as bleomycin sulfate, etoposide and cisplatin were collected from LKT Laboratories (St Paul, Minnesota).

Techniques: Derivative Assay, Cell Culture, Transplantation Assay