Journal: iScience

Article Title: Tomatidine targets ATF4-dependent signaling and induces ferroptosis to limit pancreatic cancer progression

doi: 10.1016/j.isci.2023.107408

Figure Lengend Snippet: RNA sequencing reveals that tomatidine modulates ATF4-dependent ER stress genes in pancreatic cancer cells Human and murine PDAC cell lines were treated for 40 h with 6.4 μg/mL tomatidine, RNA was isolated, and RNA sequencing was performed to analyze differences in gene regulation. N = 3 biological separate experiments. (A–D) (A) Heatmap of all genes and how they change in tomatidine-treated vs. untreated cells for Panc1 cells (FDR<0.05). Volcano plot highlighting ER stress-related genes in (B) Panc1 and (C) MT5 cells. (Upregulated genes on the right of the central axis and vice versa with higher fold change as we go away from the origin on y axis) (D) IPA upstream analysis of ATF4-related genes in Panc1 cells. (E) Heatmap elucidating targeting of ATF4-related genes in treated vs. untreated Panc1 cells. (Fold change: +2.5 to −1.5; FDR<0.03). (F) Top 10 pathways focusing on UPR, ATF4, and ER stress via Reactome analysis of the RNA-sequencing data of treated vs. untreated samples for Panc1 cells. (FDR< 0.03).

Article Snippet: The cDNA was evaluated for ATF4 and eIF4EBP1 mRNA expression using TaqMan Universal Master Mix II, no UNG (Life Technologies, Carlsbad, CA) and Taqman Gene expression assay primers for ATF4 (Hs00909569_g1), and eIF4EBP1 (Hs00607050_m1).

Techniques: RNA Sequencing, Isolation

Journal: iScience

Article Title: Tomatidine targets ATF4-dependent signaling and induces ferroptosis to limit pancreatic cancer progression

doi: 10.1016/j.isci.2023.107408

Figure Lengend Snippet: ATF4 expression in PDAC (A) KM plotter was utilized to examine ATF4 expression in human PDAC specimens examining high vs. low expression compared to overall survival. (B) Data distribution elucidating significance of the overall survival curve prepared by KM Plotter. (C and D) (C) scRNA-seq datasets of pancreatic cancer tissue from metastatic patients were obtained from NIH dbGAP (accession phs002045.v1.p1) and (D) analyzed for ATF4 expression. (See also Table S1 ). (E) Pancreatic tumor tissue from KPC mice were stained by multiplex IF and imaged using Akoya Vectra Polaris and the Phenochart software to analyze ATF4 in multiple cellular compartments. Markers used to determine ATF4 (white), epithelial cells (CK19; red), stroma (αSMA; orange and PDGFRβ; Green), CD8 T+ cells (yellow), and macrophages (F4/80; turquoise). Scale bar = 100 μm.

Article Snippet: The cDNA was evaluated for ATF4 and eIF4EBP1 mRNA expression using TaqMan Universal Master Mix II, no UNG (Life Technologies, Carlsbad, CA) and Taqman Gene expression assay primers for ATF4 (Hs00909569_g1), and eIF4EBP1 (Hs00607050_m1).

Techniques: Expressing, Staining, Multiplex Assay, Software

Journal: iScience

Article Title: Tomatidine targets ATF4-dependent signaling and induces ferroptosis to limit pancreatic cancer progression

doi: 10.1016/j.isci.2023.107408

Figure Lengend Snippet: Tomatidine inhibits ATF4-dependent signaling in PDAC (A and B) (A) MiaPaca-2 tumor cells were treated with tomatidine for 72 h and cell lysates were immunoblotted for ATF4, 4EBP1, and phospho-4EBP1(p-4EBP1) protein expression and (B) p-4EBP1/4EBP1 levels quantified by densitometry. (C) Immunofluorescence (IF) was performed on vehicle (DMSO) or tomatidine-treated Panc1 cells to track ATF4 (FITC-Green) translocation from nucleus (DAPI-Blue) to cytoplasm. Scale bar = 50 μm. (D–G) (D) Nuclear to cytoplasmic translocation was quantified. Panc1 cells were treated with vehicle (DMSO) or tomatidine for 40 h and ATF4 transcriptional activity was analyzed by chromatin immunoprecipitation (ChIP) qPCR evaluating binding of ATF4 to the downstream promoter regions of (E) eIF4EBP1 (F) CHOP- B site and (G) ASNS. Data are reported as the means + SEMs. n = 3 or more independent biological replicates (4B, One-way ANOVA with Tukey’s test for pairwise comparisons was used to analyze the data; 4D-G, Two-tailed independent student’s test was used to analyze the data, ∗p < 0.05).

Article Snippet: The cDNA was evaluated for ATF4 and eIF4EBP1 mRNA expression using TaqMan Universal Master Mix II, no UNG (Life Technologies, Carlsbad, CA) and Taqman Gene expression assay primers for ATF4 (Hs00909569_g1), and eIF4EBP1 (Hs00607050_m1).

Techniques: Expressing, Immunofluorescence, Translocation Assay, Activity Assay, Chromatin Immunoprecipitation, ChIP-qPCR, Binding Assay, Two Tailed Test

Journal: iScience

Article Title: Tomatidine targets ATF4-dependent signaling and induces ferroptosis to limit pancreatic cancer progression

doi: 10.1016/j.isci.2023.107408

Figure Lengend Snippet: In vivo tomatidine treatment inhibits pancreatic tumor growth (A–C) (A) MT5 tumor-bearing C57BL/6 mice (5 mice/group) were treated with 5 mg/kg daily i.p. injections of tomatidine or vehicle control (40% HPBCD) and monitored for tumor growth. (n = 5/group). RNA isolated from the tumor tissues of MT5 tumor-bearing C57BL/6 mice treated with vehicle or 5 mg/kg daily i.p. injections of tomatidine were assessed for (B) ATF4 and (C) eIF4EBP1 expression via qPCR. Data are reported as the means + SEMs. n = 5 mice per group. (5A, mixed between-within subjects ANOVA shows a significant interaction between days of treatment and group; 5B-C, Two-tailed independent student’s test was used to analyze the data, ∗p < 0.05).

Article Snippet: The cDNA was evaluated for ATF4 and eIF4EBP1 mRNA expression using TaqMan Universal Master Mix II, no UNG (Life Technologies, Carlsbad, CA) and Taqman Gene expression assay primers for ATF4 (Hs00909569_g1), and eIF4EBP1 (Hs00607050_m1).

Techniques: In Vivo, Control, Isolation, Expressing, Two Tailed Test

Journal: iScience

Article Title: Tomatidine targets ATF4-dependent signaling and induces ferroptosis to limit pancreatic cancer progression

doi: 10.1016/j.isci.2023.107408

Figure Lengend Snippet: Tomatidine mediated inhibition of ATF4 signaling can increase sensitivity to ferroptotic cell death in PDAC (A) Panc-1 tumor cells were treated with tomatidine (6.4 μg/mL) and analyzed by RNA sequencing. Ingenuity pathway analysis of the regulated genes suggested ferroptosis as a top hit for tomatidine-treated cells. (B–D) (B) Pancreatic cancer cells were treated with vehicle (DMSO), erastin (to induce ferroptosis), ferrostatin-1 (to inhibit ferroptosis), tomatidine, or in different combinations and lipid peroxidation of (C) Panc-1 and (D) MiaPaca-2 was analyzed by flow cytometry using Bodipy-11. (E) Panc-1 and MiaPaca-2 cells treated with vehicle (DMSO) or tomatidine and lysates collected after 24 h were immunoblot for GPX4 expression. (F) Panc1 cells were plated overnight and treated with tomatidine for 6 h and then assayed by Seahorse assay to analyze mitochondrial fitness. (G) Schematic showing how tomatidine can regulate ATF4-dependent signaling to induce ferroptosis in pancreatic cancer. Data are reported as the means + SEMs. n = 3 or more independent biological replicates. (7C-D, Two-tailed independent student’s test was used to analyze the data; 7F, Two-tailed independent student’s test was used to analyze the data, ∗p < 0.05, ∗∗p < 0.007).

Article Snippet: The cDNA was evaluated for ATF4 and eIF4EBP1 mRNA expression using TaqMan Universal Master Mix II, no UNG (Life Technologies, Carlsbad, CA) and Taqman Gene expression assay primers for ATF4 (Hs00909569_g1), and eIF4EBP1 (Hs00607050_m1).

Techniques: Inhibition, RNA Sequencing, Flow Cytometry, Western Blot, Expressing, Two Tailed Test

Journal: iScience

Article Title: Tomatidine targets ATF4-dependent signaling and induces ferroptosis to limit pancreatic cancer progression

doi: 10.1016/j.isci.2023.107408

Figure Lengend Snippet:

Article Snippet: The cDNA was evaluated for ATF4 and eIF4EBP1 mRNA expression using TaqMan Universal Master Mix II, no UNG (Life Technologies, Carlsbad, CA) and Taqman Gene expression assay primers for ATF4 (Hs00909569_g1), and eIF4EBP1 (Hs00607050_m1).

Techniques: Control, Recombinant, Viability Assay, Luciferase, ChIP-qPCR, Gene Expression, Software

Journal: Frontiers in Plant Science

Article Title: CRISPR-Cas9: Tool for Qualitative and Quantitative Plant Genome Editing

doi: 10.3389/fpls.2016.01740

Figure Lengend Snippet: Specific commercial products and services available to the researchers to implement CRISPR technology.

Article Snippet: Integrated DNA technologies (IDT) , Human HPRT PCR Primer Mix, Mouse HPRT PCR Primer Mix, Nuclease Free Duplex Buffer , S.p. Cas9 Expression Plasmid , S.p. Cas9 Nuclease 3NLS (100, 500 μg) , CRISPR Negative Control crRNA, CRISPR Positive Control crRNA.

Techniques: CRISPR, Genome Wide, Clone Assay, Stable Transfection, Selection, Transfection, Plasmid Preparation, Mutagenesis, Expressing, Negative Control, Positive Control, Construct, Knock-In, Multiplex Assay, Amplification, Sequencing