Journal: Antioxidants

Article Title: Inhibition of GCN2 Alleviates Cardiomyopathy in Type 2 Diabetic Mice via Attenuating Lipotoxicity and Oxidative Stress

doi: 10.3390/antiox11071379

Figure Lengend Snippet: GCN2iB improves cardiac function and attenuates myocardial fibrosis in type 2 diabetic (T2D) mice. High-fat diet plus low-dose streptozotocin (STZ)-induced male type 2 diabetic mice were treated with oil or GCN2iB (3 mg/kg) every other day via intraperitoneal injection for 6 weeks. At the end of the experiments, fasting blood glucose levels ( A ), body weight ( B ), heart weight ( C ), the ratio of heart weight to body weight ( D ) and left ventricular ejection fraction ( E ) were measured. ( F ) Representative heart sections were stained with wheat germ agglutinin (WGA, upper panel, scale bar = 20 μm) and Masson trichrome stain (lower panel, scale bar = 100 μm). The myocyte cross-sectional area ( G ) and myocardial fibrosis ( H ) were quantified. ( I ) The mRNA levels of hypertrophic and fibrotic genes were measured by real-time qPCR. ( J ) Heart lysates were subjected to Western blotting to measure the expression of ANP. In Figure ( A – I ), N = 5; in Figure ( J ), N = 3; values are expressed as the means ± SD; * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001.

Article Snippet: GCN2iB was purchased from MedChemExpress LLC (#HY-112654, Monmouth-Junction, NJ, USA).

Techniques: Injection, Staining, Western Blot, Expressing

Journal: Antioxidants

Article Title: Inhibition of GCN2 Alleviates Cardiomyopathy in Type 2 Diabetic Mice via Attenuating Lipotoxicity and Oxidative Stress

doi: 10.3390/antiox11071379

Figure Lengend Snippet: Effect of GCN2iB treatment on myocardial metabolomic profiles in T2D mice. ( A ) Orthogonal projections to latent structures discriminant analysis (OPLS-DA) models for the oil- and GCN2iB-treated groups are presented as score scatter plots. ( B ) The changes in myocardial metabolites in T2D mice are presented in a volcano plot. ( C ) A pie chart is used to illustrate the percentages of major metabolites of T2D mice. ( D ) KEGG pathway enrichment analysis of significantly different metabolites. ( E ) The significantly changed amino acid- and lipid-related metabolites caused by GCN2iB treatment are presented as a heatmap.

Article Snippet: GCN2iB was purchased from MedChemExpress LLC (#HY-112654, Monmouth-Junction, NJ, USA).

Techniques:

Journal: Antioxidants

Article Title: Inhibition of GCN2 Alleviates Cardiomyopathy in Type 2 Diabetic Mice via Attenuating Lipotoxicity and Oxidative Stress

doi: 10.3390/antiox11071379

Figure Lengend Snippet: GCN2iB affects gene expression profiles in T2D mice. ( A ) A volcano plot was used to show the fold changes in differentially expressed genes (DEGs) in the control group vs. the GCN2iB group. ( B ) Twelve significantly enriched KEGG pathways are listed as an advanced bubble chart. ( C ) The gene expression profiles of the DEGs that were involved in oxidative phosphorylation, the PPAR signaling pathway and glycolysis/gluconeogenesis pathways are shown in the heatmap. ( D , E ) The gene expression profiles of the DEGs that were involved in cardiac function, proteasome and biosynthesis of amino acids pathways are shown in the heatmap. ( F ) The mRNA levels of some randomly selected differentially expressed genes were measured by qPCR. In Figure ( F ), N = 5, values are expressed as the means ± SD; * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001.

Article Snippet: GCN2iB was purchased from MedChemExpress LLC (#HY-112654, Monmouth-Junction, NJ, USA).

Techniques: Expressing, Control

Journal: Antioxidants

Article Title: Inhibition of GCN2 Alleviates Cardiomyopathy in Type 2 Diabetic Mice via Attenuating Lipotoxicity and Oxidative Stress

doi: 10.3390/antiox11071379

Figure Lengend Snippet: GCN2iB ameliorates myocardial lipid accumulation and oxidative stress in T2D mice. ( A ) Cardiac triglyceride (TG) levels were measured in oil- and GCN2iB-treated T2D mice. ( B ) The mRNA levels of lipid metabolism-related genes were measured. ( C , D ) Cardiac 3′-nitrotyrosine (3′-NT) and 4-hydroxynonenal (4-HNE) levels were measured. ( E ) Representative heart sections were stained with dihydroethidium (DHE), and the relative fluorescence intensity was quantified. Scale bar = 50 μm. ( F ) The expression profiles of some antioxidative genes are displayed as a heatmap. ( G ) Heart lysates were examined by Western blot. In Figure ( A – E ), N = 5; in ( F , G ), N = 3; values are expressed as the means ± SD; * indicates p < 0.05; ** indicates p < 0.01.

Article Snippet: GCN2iB was purchased from MedChemExpress LLC (#HY-112654, Monmouth-Junction, NJ, USA).

Techniques: Staining, Fluorescence, Expressing, Western Blot

Journal: Antioxidants

Article Title: Inhibition of GCN2 Alleviates Cardiomyopathy in Type 2 Diabetic Mice via Attenuating Lipotoxicity and Oxidative Stress

doi: 10.3390/antiox11071379

Figure Lengend Snippet: GCN2iB improves cardiac function and alleviates myocardial oxidative stress and lipid accumulation in db/db mice. Db/db mice were treated with GCN2iB (3 mg/kg) every other day via intraperitoneal injection for 6 weeks. Then, fasting blood glucose levels ( A ), body weight ( B ), heart weight ( C ), ratio of heart weight to body weight ( D ), left EF ( E ), cardiac TG levels ( F ), 3′-NT levels ( G ) and 4-HNE levels ( H ) were measured. ( I ) The mRNA levels of genes related to hypertrophy, fibrosis, lipid metabolism and oxidative phosphorylation were measured. ( J ) Heart lysates were examined by Western blot. ( K ) Schematic diagram shows the potential target cell affected by GCN2iB in T2D mice. In Figure ( A – I ), N = 5; in Figure ( J ), N=3; values are expressed as the means ± SD; * indicates p < 0.05; ** indicates p < 0.01.

Article Snippet: GCN2iB was purchased from MedChemExpress LLC (#HY-112654, Monmouth-Junction, NJ, USA).

Techniques: Injection, Western Blot

Journal: The EMBO Journal

Article Title: The integrated stress response drives MET oncogene overexpression in cancers

doi: 10.1038/s44318-024-00338-4

Figure Lengend Snippet: ( A ) Schematic overview of translational control under steady-state and microenvironmental stress conditions. The diagram illustrates the transition from high levels of cap-dependent translation to preferential translation of stress-responsive genes via the integrated stress response-inducing eIF2α phosphorylation (Pakos-Zebrucka et al, ). The schematic also shows the action of three ISR inhibitors: GCN2 inhibitor GCN2iB (Nakamura et al, ), PER K inhibitor GSK2656157 (Atkins et al, ), and ISRIB (Rabouw et al, ; Zyryanova et al, ). ( B ) Western blot analysis showing the protein levels of MET, eIF2α-P, and ATF4 in cells subjected to 24 h of SS and treated with increasing concentrations of salubrinal (1, 5, and 10 µM). In control conditions, cells were grown in the presence of 10% serum without salubrinal. TBP was used as a loading control. ( C ) RT-qPCR analysis of MET and ATF4 mRNA levels in cells treated as described in ( B ). mRNA levels are presented as fold change relative to the control condition, normalised to TBP expression. Error bars represent mean ± SEM ( n = 3 biological replicates), with statistical analysis performed using one-way ANOVA and Tukey’s post hoc test. ( D ) Western blot analysis comparing cells expressing the wild-type eIF2α or in CRISPR/Cas9 cells expressing non-phosphorylatable eIF2α S52A mutant. Phosphorylated eIF2α (eIF2α-P), ATF4, MET, phosphorylated MET (MET-P), and phosphorylated AKT (AKT-P) levels under normal and serum starvation (SS) conditions ±HGF (50 ng/mL), using TBP as a loading control. ( E ) Total RNA was extracted to assess MET and ATF4 expression levels by RT-qPCR using TBP as a normalisation gene. Error bars represent mean ± SEM ( n = 3 biological replicates), with statistical analysis performed using one-way ANOVA and Tukey’s post hoc test. ( F ) Cell viability assay showing the proliferation profiles of cells with indicated genotypes, illustrating the impact of the eIF2α S52A mutation on cell survival during serum starvation. Cells were treated with or without 50 ng/mL HGF, and outcomes were normalised to fluorescence readings at t = 1 day. Error bars represent SEM ( n > 6), and data were analysed using two-way ANOVA with repeated measures. ( G ) Dose–response experiments on cells with the indicated genotype to increasing concentrations of GEM ± 50 ng/mL HGF for 48 h. Results were standardised to untreated conditions in wt cells, and IC 50 values are shown in the inset. Error bars represent SEM ( n = 3 biological replicates), with statistical analysis performed using one-way ANOVA and Tukey’s post hoc test. Significance markers: **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, and “ns” indicates not significant. .

Article Snippet: GCN2iB , Selleckchem , S8929.

Techniques: Control, Phospho-proteomics, Western Blot, Quantitative RT-PCR, Expressing, CRISPR, Mutagenesis, Viability Assay, Fluorescence

Journal: The EMBO Journal

Article Title: The integrated stress response drives MET oncogene overexpression in cancers

doi: 10.1038/s44318-024-00338-4

Figure Lengend Snippet: ( A ) Western blot analysis of EKVX cells cultured with varying serum concentrations ± 500 nM GCN2iB for 24 h, probing for MET, eIF2α-P, ATF4, and TBP (loading control). Heatmaps depict the relative intensities of bands, where yellow highlights low levels, and blue indicates high levels. ( B ) Evaluation of MET mRNA expression by RT-qPCR in cells subjected to different serum conditions ± GCN2iB, normalised to TBP mRNA levels. Error bars represent SEM ( n = 3 biological replicates). Statistical analysis was performed using two-way ANOVA. ( C ) Western blot analysis of EKVX cells treated with 500 nM Tg for 6 h. Where indicated, cells were pre-treated with increasing concentrations of the PERK inhibitor GSK2656157 for 1 h before Tg addition. Cells were probed for MET, phosphorylated eIF2α (eIF2α-P), ATF4, and TBP (loading control). The heatmaps below the blots represent the relative band intensities. ( D ) RT-qPCR analysis of MET mRNA levels in EKVX cells treated with 500 nM Tg for 24 h in the presence or absence of the PERK inhibitor GSK2656157 (GSK, 10 nM, 100 nM, or 1000 nM). mRNA levels were normalised to TBP mRNA levels, and data are presented as the mean ± SEM ( n = 3 biological replicates). Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test. ( E ) The impact of ISR inhibition by ISRIB (0; 50 nM; 500 nM; or 5000 nM) on eIF2α phosphorylation, ATF4, and MET levels under 24 h SS conditions, compared to the control and GCN2iB treatment (500 nM), with TBP as the loading control. ( F ) RT-qPCR analysis of MET mRNA levels in EKVX cells under 24 h serum starvation conditions treated with ISRIB (0, 50 nM, 500 nM, or 5000 nM) or GCN2iB (500 nM). MET mRNA levels were normalised to TBP mRNA levels, and data are presented as the mean ± SEM. With n = 3 biological replicates. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test. ( G ) Protein expression analysis via western blot in a panel of cell lines cultured with or without serum and treated with ±GCN2iB (500 nM) for 24 h. ( H ) Assessment of MET mRNA levels and MET gene copy number using the CCLE database (Barretina et al, ). ( I ) Mutational status (blue squares indicate mutations) and copy number variations (CNV) in various cancer driver genes based on the CCLE database (Barretina et al, ). Significance marker: **** P < 0.0001. .

Article Snippet: GCN2iB , Selleckchem , S8929.

Techniques: Western Blot, Cell Culture, Control, Expressing, Quantitative RT-PCR, Inhibition, Phospho-proteomics, Marker

Journal: The EMBO Journal

Article Title: The integrated stress response drives MET oncogene overexpression in cancers

doi: 10.1038/s44318-024-00338-4

Figure Lengend Snippet: ( A ) RT-qPCR analysis of ATF4 , DDIT3 , ATF3 , and GADD45A in EKVX cells under control condition (10% serum) or decreasing amount of serum (from 5% to 0%) ± 500 nM GCN2iB. The graphs depict fold changes over control with TBP as a reference gene. Error bars represent SEM with n = 3 biological replicates. Data were analysed using two-way ANOVA. ( B ) Cells were cultured in 10% serum (control) or under serum starvation ± increasing concentrations of GCN2iB for 24 h and treated with ± 50 ng/mL of HGF for 15 min. Western blot panels showing MET, MET-P, AKT-P, eIF2α-P, and ATF4 levels, with TBP as the loading control. ( C ) RT-qPCR analysis of ATF4 , DDIT3 , ATF3 , and GADD45A mRNA levels in EKVX cells treated with 500 nM Tg for 24 h in the presence or absence of the PERK inhibitor GSK2656157 (GSK, 10 nM, 100 nM, or 1000 nM). mRNA levels were normalised to TBP , and results are presented as fold changes over control conditions. Error bars represent SEM with n = 3 biological replicates. Data were analysed by one-way ANOVA with Tukey’s post hoc test. ( D ) RT-qPCR experiments showing MET mRNA expression across a panel of cell lines under control condition (10% serum) or serum starvation (0% serum) ± 500 nM GCN2iB for 24 h. Results are fold change over control condition normalised with TBP . Results are presented as mean ± SEM with n = 3 biological replicate and analysed using two-way ANOVA. ( E ) The viability of EKVX cells is quantified over six days under control condition (10% Serum) ± HGF (50 ng/mL) ± ISR inhibitors GCN2iB or ISRIB. Error bars represent SEM with n > 3 biological replicates. Two-way ANOVA with repeated measures was used to evaluate significance (non-significant). Significance is denoted as **** P < 0.0001, *** P < 0.001, and ** P < 0.01.

Article Snippet: GCN2iB , Selleckchem , S8929.

Techniques: Quantitative RT-PCR, Control, Cell Culture, Western Blot, Expressing

Journal: The EMBO Journal

Article Title: The integrated stress response drives MET oncogene overexpression in cancers

doi: 10.1038/s44318-024-00338-4

Figure Lengend Snippet: ( A ) Heatmap visualisation of EMT marker expression profiles obtained by RT-qPCR in EKVX cells cultured under various conditions for 72 h. Red indicates upregulation, and blue indicates downregulation of gene expression normalised to TBP expression. ( B ) Immunofluorescence staining of EKVX cells for E-Cadherin (epithelial marker, magenta) and ACTA2 (mesenchymal marker, yellow) under control (10% serum) or SS for 72 h ± 500 nM GCN2iB ± 50 ng/mL HGF. Nuclei counterstained with DAPI (cyan). Scale bar: 25 µm. Fluorescence intensity was quantified using ImageJ software from n > 3 biological replicates. Two-way ANOVA with repeated measures was used for statistical analysis. Represented P values: SS vs. SS + HGF: 4.11 × 10 −4 (****); SS + HGF vs. SS + HGF + GCN2iB: 6.03 × 10 −4 (****); SS - HGF + GCN2iB vs. SS + HGF + GCN2iB: 0.054 (“ns”); SS vs. SS - HGF + GCN2iB: 0.25 (“ns”); + serum - HGF vs. + serum + HGF: 0.065 (“ns”); SS + HGF vs. + serum + HGF: 2.1 × 10 −5 (****); SS - HGF vs. + serum - HGF: 0.856 (“ns”). ( C ) Kinetic analysis of wound healing in EKVX cells under serum starvation conditions ± 500 nM GCN2iB ± 50 ng/mL HGF. Wound density was monitored every 2 h. Data presented as relative wound density (%) are mean ± SEM, with n = 6 biological replicates. Results were analysed with two-way ANOVA with repeated measures. Represented p values for control vs. +HGF comparisons at the end of the experiment: P = 2.22 × 10 −4 (***). ( D ) Viability of EKVX cells measured over time under serum starvation ± 50 ng/ml HGF ± 500 nM GCN2iB ± 1 µM ISRIB. Results are normalised to initial viability (day 1) and are represented as mean ± SEM with n = 6 biological replicates. Data were analysed using two-way ANOVA with repeated measures. Represented P values for control vs. +HGF comparisons: day 1: 0.2493 (ns); day 2: 0.1302 (ns); day 3: 3.5 × 10 − 3 (**); day 4: 2.2 × 10 − 3 (**). Comparisons of +HGF + ISRIB vs. control and +HGF + GCN2iB vs. control remained “ns” throughout the experiment. ( E ) Western blot analysis of MET, ATF4, and eIF2α-P levels in EKVX cells after treatment with increasing GEM concentrations (5 nM; 50 nM; 500 nM). For ISR inhibition (with either 500 nM GCN2iB or 1 µM ISRIB), cells were treated with 500 nM of GEM. TBP is used as a loading control. ( F ) Dose–response survival curves of EKVX cells treated with GEM for 48 h across a range of concentrations, with or without additional treatments (50 ng/mL HGF, 500 nM GCN2iB, or 1 µM ISRIB). The inset shows IC 50 quantifications for each treatment condition. Results are presented as mean ± SEM ( n > 3 biological replicates) and analysed using one-way ANOVA with Tukey’s post hoc test. Significance markers: ns (not significant), ** P < 0.01, *** P < 0.001, **** P < 0.0001. .

Article Snippet: GCN2iB , Selleckchem , S8929.

Techniques: Marker, Expressing, Quantitative RT-PCR, Cell Culture, Gene Expression, Immunofluorescence, Staining, Control, Fluorescence, Software, Western Blot, Inhibition

Journal: The EMBO Journal

Article Title: The integrated stress response drives MET oncogene overexpression in cancers

doi: 10.1038/s44318-024-00338-4

Figure Lengend Snippet: Reagents and tools table

Article Snippet: GCN2iB , Selleckchem , S8929.

Techniques: Recombinant, Sequencing, Transfection, Western Blot, Luciferase, CyQUANT Assay, Proliferation Assay, Plasmid Preparation, Software

Journal: eLife

Article Title: Distinct mitochondrial defects trigger the integrated stress response depending on the metabolic state of the cell

doi: 10.7554/eLife.49178

Figure Lengend Snippet: ( A ) Secreted [lactate] following 2 hr piericidin treatment in control (Ctrl), Lb NOX ( Lb ) or NDI1 cells. Data is normalized to DMSO (-) separately for each cell line. Mean ± SD, N = 6 from three experiments (same samples as in ). Welch’s t-test (two-tailed) was used to compare control with Lb NOX and NDI1 cells, followed by Holm’s correction for multiple testing. ( B ) Adenylate energy charge following 1 hr piericidin treatment in control, Lb NOX or NDI1 cells. Mean ± SD, N = 5–6 from two experiments. The Games-Howell test was used to make all pairwise comparisons. Notations with no connecting lines relate to the equivalent treatment in control cells. ( C ) Fold-change (x-axis) and statistical significance (y-axis) of metabolite differential abundance following 1 hr piericidin treatment in extracts of control or Lb NOX cells. N = 3. P-Ser, phosphoserine; Asp, aspartate; Asn, asparagine; Cit, citrate; Suc, succinate; αKG, α-ketoglutarate; IMP, inosine monophosphate; AICAR, 5-aminoimidazole-4-carboxamide ribonucleotide; R5P, ribose 5-phosphate; G3P/DHAP, glyceraldehyde 3-phosphate and/or dihydroxyacetone phosphate; P-Cr, phosphocreatine; Pro, proline. Detailed results are provided in . ( D ) Intracellular [aspartate] following 1 hr piericidin treatment in control or Lb NOX cells. Data is normalized to DMSO in control cells. Mean ± SD, N = 6 from two experiments (includes samples shown in C). The Games-Howell test was used for all pairwise comparisons. ( E ) Peak intensity of intracellular asparagine following 1 hr piericidin treatment in control cells, with or without aspartate, and in Lb NOX cells. Data is normalized to DMSO in control cells. Mean ± SD, N = 6 from two experiments. The Games-Howell test was used for all pairwise comparisons. ( F ) qPCR of Ddit3 following 10 hr piericidin treatment in control cells, with or without aspartate, and in Lb NOX cells. Data is presented as fold-change from DMSO in control cells. Mean ± SD, N = 8 from three experiments. The Games-Howell test was used to make all pairwise comparisons of ΔΔC t values. ( G ) Media [lactate]/[pyruvate] following 2 hr piericidin treatment, with or without aspartate, in control cells. Data is normalized to DMSO without aspartate. Mean ± SD, N = 6 from three experiments. Welch’s t-test (two-tailed) was used to compare each treatment with and without aspartate, followed by Holm’s correction. ( H ) Proliferative rate (doublings in 24 hr) of control cells, with or without aspartate, and of Lb NOX cells following piericidin treatment. Data is normalized to DMSO in control cells. Mean ± SD, N = 5–6 from three experiments. The Games-Howell test was used for all pairwise comparisons. ( I ) qPCR of Atf3 following 10 hr pyruvate withdrawal, with or without aspartate, in Ndufa9 -KO C2C12 myoblasts. Data is presented as fold-change from the condition with pyruvate (+). Mean ± SD, N = 3. ( J ) qPCR of GDF15 following 10 hr piericidin treatment, with or without pyruvate or aspartate, in primary human skeletal myoblasts. Data is presented as fold-change from DMSO. Mean ± SD, N = 3. ( K ) qPCR of Atf3 following 10 hr piericidin or tunicamycin (Tuni) treatment, with or without GCN2iB, in control cells. Data is presented as fold-change from DMSO. Mean ± SD, N = 6-7. Welch’s t-test (two-tailed) was used to compare each treatment with and without GCN2iB, followed by Holm’s correction. ( L ) Western blot of (p-)GCN2, ATF4 and (p-)eIF2α following 6 hr piericidin treatment in the indicated conditions in Lb NOX cells. Lb NOX expression was induced only where indicated. ( M ) qPCR of Atf3 and Gdf15 in the same cells and conditions shown in L. Data is presented as fold-change from DMSO. Mean ± SD, N = 2–3. GiB, GCN2iB. ( N ) Model for ISR activation by complex I inhibition in myoblasts. ns, p>0.05; *, p<0.05; **, p<0.01; ***, p<0.001. Figure 4—source data 1. Metabolite profiling data.

Article Snippet: Chemical compound, drug , GCN2iB , Acme Bioscience (PMID: 30061420 ) , , 0.5 μM.

Techniques: Control, Two Tailed Test, Western Blot, Expressing, Activation Assay, Inhibition

Journal: eLife

Article Title: Distinct mitochondrial defects trigger the integrated stress response depending on the metabolic state of the cell

doi: 10.7554/eLife.49178

Figure Lengend Snippet: ( A ) Secreted [lactate] following 2 hr inhibitor treatments in control or Lb NOX cells. Data is normalized to DMSO separately in each cell line. Mean ± SD, N = 6 from three experiments (expanded version of ). ( B ) Adenylate energy charge following 1 hr treatments in control or Lb NOX cells. Mean ± SD, N = 5–6 from two experiments (expanded version of ). ( C ) Scatter plot of intracellular metabolite fold-changes between piericidin and DMSO in control cells (x-axis) or Lb NOX cells (y-axis). N = 3 replicates (same data as in ). ( D ) Intracellular [aspartate] following 1 hr treatments in control or Lb NOX cells. Mean ± SD, N = 6 from two experiments (expanded version of ). ( E ) Peak intensity of intracellular asparagine following 1 hr treatments in control or Lb NOX cells. Mean ± SD, N = 6 from two experiments (expanded version of ). ( F )-( I ) qPCR of ISR-related transcripts following 10 hr treatments, with or without aspartate, in control cells. Data is presented as fold-change from DMSO without aspartate. Mean ± SD, N = 3. Ppp1r15a , protein phosphatase 1 regulatory subunit 15A. ( J ) qPCR of Atf3 following 10 hr piericidin treatment, with or without pyruvate or aspartate, in primary mouse embryonic fibroblasts. Data is presented as fold-change from DMSO (-). Mean ± SD, N = 3. ( K ) qPCR of Atf3 following 10 hr piericidin or tunicamycin treatment, with or without aspartate, GCN2iB or the PERK inhibitor GSK2656157, in control cells. Data is presented as fold-change from DMSO. Mean ± SD, N = 3. ( L ) Ratio of p-eIF2α to total eIF2α, measured by western blot, following 6 hr piericidin treatment in Lb NOX cells. Lb NOX expression was induced only where indicated. Data is normalized separately in each blot to the DMSO-treated sample not expressing Lb NOX. Mean ± SD, N = 4 from four experiments. A one-sample t-test was used to examine whether the mean of DMSO-treated samples expressing Lb NOX differed from 1, and Welch’s t-test (two-tailed, paired by blot) was used to compare the piericidin-treated samples with and without Lb NOX expression. Holm’s correction was then applied. ns, p>0.05; **, p<0.01.

Article Snippet: Chemical compound, drug , GCN2iB , Acme Bioscience (PMID: 30061420 ) , , 0.5 μM.

Techniques: Control, Western Blot, Expressing, Two Tailed Test

Journal: eLife

Article Title: Distinct mitochondrial defects trigger the integrated stress response depending on the metabolic state of the cell

doi: 10.7554/eLife.49178

Figure Lengend Snippet:

Article Snippet: Chemical compound, drug , GCN2iB , Acme Bioscience (PMID: 30061420 ) , , 0.5 μM.

Techniques: Knock-Out, Recombinant, Luciferase, Expressing, Plasmid Preparation, Modification, Concentration Assay, Gene Expression

Journal: Cancer Gene Therapy

Article Title: Arsenic trioxide and p97 inhibitor synergize against acute myeloid leukemia by targeting nascent polypeptides and activating the ZAKα–JNK pathway

doi: 10.1038/s41417-024-00818-z

Figure Lengend Snippet: A The NB4 cells were pre-treated with 0.1 μg/mL puromycin for 1 h, and then 4 μM Biotin-As was added for another 16 h. Immunofluorescence visualized the localization of puromycin, Biotin-As, and aggresome in the cells. Scale bar, 25 μm. B The NB4 cells were treated with 1 μM Biotin-As for 4 h, and then 20 μM puromycin was added for another 30 min. Cell fractions were prepared for the Co-IP assays using the streptavidin magnetic beads and indicated antibodies. C Immunoblot of indicated proteins in the NB4 cells at 0, 4, 8, 12, and 24 h with 0.5 or 2 μM ATO treatment. D The NB4 cells were pre-treated with 0.5 μM PERKi (GSK2656157) or 10 μM GCN2iB for 1 h, and then 2 μM ATO was added for another 4 h. Immunoblot of indicated proteins in the NB4 cells.

Article Snippet: Arsenic trioxide, N-acetyl-L-cysteine and H 2 O 2 (Sigma, St Louis, MO, USA); Glutathione, DCFH-DA (Beyotime Biotechnology, Shanghai, China); CCK-8, GCN2iB, NMS873, JNK-IN-8 (Topscience, Shanghai, China); Anisomycin, puromycin, rapamycin, cycloheximide, thapsigargin, tunicamycin, 4EGI-1 (MedChemExpress, Monmouth, NJ, USA); GSK2656157 (Selleck, Houston, TX, USA).

Techniques: Immunofluorescence, Co-Immunoprecipitation Assay, Magnetic Beads, Western Blot

Journal: eLife

Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

doi: 10.7554/eLife.81083

Figure Lengend Snippet: ( A ) Expression of the indicated eIF2α kinase was reduced in LNCaP cells using gene-specific siRNAs. Two different siRNAs were used for knockdown of each eIF2α kinase and compared to scrambled siRNA control. Cell growth was measured in replicate wells ( N = 5) for up to 6 days and is plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; *p ≤ 0.05, ***p ≤ 0.001, ****p ≤ 0.0001. ( B ) LNCaP cells were transfected with two different siRNAs targeting GCN2 or a scramble siRNA control and cell lysates were prepared and immunoblotted for the indicated proteins. Molecular weight markers are shown in kilodaltons. The relative levels of p-eIF2α normalized to total eIF2α compared to scramble siRNA control are indicated. ( C ) Expression of GCN2 was knocked-down in LAPC-4, C4-2B, MR49F, 22Rv1, or PC-3 cells using two different siRNAs and compared to scrambled siRNA control. Cell growth was measured for up to 6 days in replicate wells ( N = 5) as described in A . Statistical significance was determined using a two-way ANOVA as described in ; ***p ≤ 0.001, ****p ≤ 0.0001. ( D ) LNCaP cells were treated with indicated concentrations of GCN2iB and cell growth was measured for up to 6 days in replicate wells ( N = 5) as described in A . Statistical significance was determined using a two-way ANOVA as described in ; ****p ≤0.0001. ( E ) LNCaP cells were treated with GCN2iB (2 µM) or DMSO control for 24 hr and protein lysates were analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2-T899, total or phosphorylated eIF2α−S51, ATF4, or actin as indicated. Relative levels of p-eIF2α normalized to total eIF2α are shown. ( F ) Levels of p-GCN2 were measured in prostate tumor microarrays (Biomax PR1921b and PR807c) using immunohistochemistry (IHC). Staining for p-GCN2-T899 from non-malignant ( N = 33) and malignant PCa tissue ( N = 88) from patients >50 years old was analyzed and quantified using QuPath to determine the histoscore and is represented as a scatterplot. Statistical significance was determined using an unpaired two-tailed t -test; *p ≤ 0.05. Representative images showing p-GCN2-T899 staining of normal and malignant prostate tissues are shown. Scale bars shown are 200 µm (main image) and 20 µm (insert).

Article Snippet: Media was changed to standard culture media containing 500 nM to 10 µM GCN2iB or DMSO as a control or standard media lacking histidine (MyBiosource, Cat. #MBS652918) and incubated for the indicated time period.

Techniques: Expressing, Standard Deviation, Transfection, Molecular Weight, Western Blot, Immunohistochemistry, Two Tailed Test, Staining

Journal: eLife

Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

doi: 10.7554/eLife.81083

Figure Lengend Snippet: ( A ) C4-2B or 22Rv1 cells, cultured as indicated in the Materials and methods, or PC-3 cells cultured in HPLM media were treated with GCN2iB as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001. ( B ) 22Rv1 WT and 22Rv1 GCN2 KO (clone 7) cells were treated with GCN2iB as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± SD) relative to day 0. Statistical significance was determined using a two-way ANOVA as described in ; ****p ≤ 0.0001. ( C ) Lysates were prepared from C4-2B, 22Rv1, or PC-3 cells treated with GCN2iB at the indicated concentrations or vehicle control (dimethyl sulfoxide, DMSO) for 48 hr and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, ASNS, TRIB3, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2) AR, or actin. Molecular weight markers are indicated in kilodaltons.

Article Snippet: Media was changed to standard culture media containing 500 nM to 10 µM GCN2iB or DMSO as a control or standard media lacking histidine (MyBiosource, Cat. #MBS652918) and incubated for the indicated time period.

Techniques: Cell Culture, Standard Deviation, Western Blot, Molecular Weight

Journal: eLife

Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

doi: 10.7554/eLife.81083

Figure Lengend Snippet: ( A ) Lysates were prepared from BPH-1, LNCaP C4-2B, 22Rv1, or PC-3 cells and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, AR, or actin. Molecular weight markers are indicated in kilodaltons. ( B ) BPH-1 cells were transfected with siRNAs targeting GCN2, ATF4, or 4F2 (SLC3A2). Protein lysates were prepared and analyzed by immunoblot to determine the levels of GCN2, ATF4, 4F2 (SLC3A2), or actin as indicated. Molecular weight markers are indicated in kilodaltons. ( C ) Expression of GCN2, ATF4, or 4F2 (SLC3A2) was reduced in BPH-1 cells using two different gene-specific siRNAs as indicated and compared to a scramble siRNA control. Cell growth was measured for up to 6 days in replicate wells ( N = 5) as described in A . Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; *p ≤ 0.05, **p ≤ 0.01. ( D ) Lysates were prepared from BPH-1 cells treated with GCN2iB at the indicated concentrations or vehicle control (DMSO) for 48 hr and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, ASNS, TRIB3, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), AR, or actin. Molecular weight markers are indicated in kilodaltons. ( E ) BPH-1 cells were treated with 0.5–10 µM GCN2iB or vehicle (DMSO) control as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way ANOVA is shown in .

Article Snippet: Media was changed to standard culture media containing 500 nM to 10 µM GCN2iB or DMSO as a control or standard media lacking histidine (MyBiosource, Cat. #MBS652918) and incubated for the indicated time period.

Techniques: Western Blot, Molecular Weight, Transfection, Expressing, Standard Deviation

Journal: eLife

Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

doi: 10.7554/eLife.81083

Figure Lengend Snippet: ( A ) Volcano plot illustrating log 2 fold change in gene transcript levels with adjusted p value (−log 10 ) comparing LNCaP cells treated with GCN2iB (2 µM) versus vehicle control (DMSO) for 24 hr. Several amino acid transporters reduced by GCN2iB treatment are highlighted. ( B ) Plots from gene set enrichment analysis (GSEA) of gene expression in LNCaP cells treated with GCN2iB (2 µM) for 24 hr versus vehicle control. ( C ) Heat map displaying significantly downregulated SLC genes as indicated in panel A . The heat map compares gene transcript levels from LNCaP cells treated with vehicle (DMSO), or GCN2iB (2 µM) for 6 or 24 hr. Four biological replicates were measured for each treatment group. Transcript levels (normalized read counts) are shown relative to the average of the vehicle control samples for each gene. ( D ) Lysates were prepared from LNCaP cells treated with 2 µM GCN2iB or vehicle control (DMSO) for 6 or 24 hr and immunoblot analysis were carried out using antibodies that recognize ATF4, ASNS, xCT (SLC7A11), 4F2 (SLC3A2), CAT1 (SLC7A1), ASCT1 (SLC1A4), ASCT2 (SLC1A5), or actin. Molecular weight markers are indicated in kilodaltons. ( E ) 22Rv1 WT cells, 22Rv1 GCN2 KO cells, and 22Rv1 GCN2 KO complemented with GCN2 cells were cultured for 24 hr. Lysates were prepared and analyzed by immunoblot for the indicated proteins. ( F ) Amino acid uptake measurements in LNCaP and 22Rv1 cells treated with vehicle (DMSO) or GCN2iB (2 µM) for 24 hr. ( G ) Amino acid uptake measurements for 22Rv1 WT or 22Rv1 GCN2 KO cells cultured for 24 hr. Statistical significance was determined using an unpaired two-tailed t -test ( N = 4); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Article Snippet: Media was changed to standard culture media containing 500 nM to 10 µM GCN2iB or DMSO as a control or standard media lacking histidine (MyBiosource, Cat. #MBS652918) and incubated for the indicated time period.

Techniques: Expressing, Western Blot, Molecular Weight, Cell Culture, Two Tailed Test

Journal: eLife

Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

doi: 10.7554/eLife.81083

Figure Lengend Snippet: ( A ) LNCaP cells were treated with 2 µM GCN2iB or vehicle control (DMSO) for 6 or 24 hr, protein lysates were prepared, and immunoblotted for the indicated proteins. The bar graphs show the relative levels of the indicated proteins normalized to actin. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD) ( N = 3); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. ( B ) Immunoblot analysis of PC-3 WT, PC-3 GCN2 KO (clone C-2), and PC-3 GCN2 KO (clone C-3) lysates using antibodies that recognize GCN2, ATF4, ASNS, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), ASNS, or actin.

Article Snippet: Media was changed to standard culture media containing 500 nM to 10 µM GCN2iB or DMSO as a control or standard media lacking histidine (MyBiosource, Cat. #MBS652918) and incubated for the indicated time period.

Techniques: Two Tailed Test, Standard Deviation, Western Blot

Journal: eLife

Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

doi: 10.7554/eLife.81083

Figure Lengend Snippet: ( A ) Amino acid measurements of LNCaP cells treated with 2 µM GCN2iB or vehicle control (DMSO) for 8 hr. Bar graphs in the top panel show high abundance amino acids and the lower panel those with lower levels. The heat map on the right shows fold change in amino acid abundance for each biological replicate of GCN2iB-treated LNCaP cells versus the vehicle with the scale showing the highest fold change in yellow and lowest in purple. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD) ( N = 3); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. ( B ) LNCaP cells were treated with vehicle, GCN2iB (2 µM), vehicle + essential amino acids (EAA), or GCN2iB (2 µM) + EAA, and cell growth was measured for up to 6 days. Error bars indicate SD ( N = 5). Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001. ( C ) Cell cycle analyses of LNCaP cells treated with vehicle, GCN2iB (2 µM), vehicle + EAA, or GCN2iB (2 µM) + EAA for 48 hr. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD ( N = 3); ***p ≤ 0.001, ****p ≤ 0.0001. ( D ) Genome-wide tRNA charging analysis (CHARGE-seq) of LNCaP cells treated with vehicle (DMSO), GCN2iB (2 µM), or GCN2iB (2 µM) + EAA for 8 hr. The tRNA charging ratio is shown as a bar graph with fold change compared to vehicle. Only tRNA isoacceptors measured in LNCaP cells are shown. Error bars indicate SD ( N = 4). ( E ) tRNA charging percentage for tRNA His in LNCaP cells treated with vehicle, GCN2iB, or GCN2iB + EAA. Statistical significance was determine using a one-way ANOVA with Tukey’s multiple comparisons ( N = 4); ***p ≤ 0.001, ****p ≤ 0.0001. ( F ) LNCaP cells were treated with vehicle, GCN2iB (2 µM), GCN2iB (2 µM) + EAA, or GCN2iB (2 µM) combined with the indicated individual amino acids. Cell growth was measured at 4 days in triplicate wells ( N = 3). Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD; ****p ≤ 0.0001. ( G ) Cell cycle analysis of LNCaP cells were treated with vehicle, GCN2iB (2 µM), GCN2iB (2 µM) + histidine (200 µM), or with media lacking histidine for 48 hr. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD ( N = 3); ***p ≤ 0.001, ****p ≤ 0.0001. ( H ) LNCaP cells were cultured in normal media, media supplemented with EAA mix, or media supplemented with histidine (200 µM) for 24 hr. Lysates were analyzed by Immunoblot using antibodies that recognize total or phosphorylated GCN2-T899, total or phosphorylated eIF2α−S51, ATF4, or actin. Molecular weight markers are presented in kilodaltons for each immunoblot panel. The relative levels of p-eIF2α normalized to total eIF2α compared to normal media (NM) control are indicated.

Article Snippet: Media was changed to standard culture media containing 500 nM to 10 µM GCN2iB or DMSO as a control or standard media lacking histidine (MyBiosource, Cat. #MBS652918) and incubated for the indicated time period.

Techniques: Two Tailed Test, Standard Deviation, Genome Wide, Cell Cycle Assay, Cell Culture, Western Blot, Molecular Weight

Journal: eLife

Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

doi: 10.7554/eLife.81083

Figure Lengend Snippet: LNCaP cells were treated with GCN2iB (2 µM) or vehicle control (DMSO) in standard growth media, media supplemented with non-essential amino acids (NEAA), supplemented with essential amino acids (EAA), or supplemented with histidine (200 µM), and cultured for up to 6 days. Cell growth was quantitated using CellTiter-Glo as described in the Materials and methods and is presented as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001.

Article Snippet: Media was changed to standard culture media containing 500 nM to 10 µM GCN2iB or DMSO as a control or standard media lacking histidine (MyBiosource, Cat. #MBS652918) and incubated for the indicated time period.

Techniques: Cell Culture, Standard Deviation

Journal: eLife

Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

doi: 10.7554/eLife.81083

Figure Lengend Snippet: ( A ) LNCaP cells were treated with 2 µM GCN2iB or vehicle (DMSO) control in the absence or presence of EAA for 8 hr and tRNA His charging levels were determined by qRT-PCR as described in the Materials and methods. ( B ) LNCaP cells were treated with GCN2iB (2 µM) or vehicle (DMSO) control in the absence or presence of histidine supplementation. Puromycin (1 µM) was added to culture media 15 min prior to lysate preparation and puromycin incorporation was measured by immunoblot analysis using anti-puromycin antibody. Puromycin incorporation was quantified from replicate samples and is shown normalized to the vehicle control levels. Statistical significance was determined using a one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons. Error bars indicate standard deviation (SD) ( N = 3); ns, p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Article Snippet: Media was changed to standard culture media containing 500 nM to 10 µM GCN2iB or DMSO as a control or standard media lacking histidine (MyBiosource, Cat. #MBS652918) and incubated for the indicated time period.

Techniques: Quantitative RT-PCR, Western Blot, Standard Deviation

Journal: eLife

Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

doi: 10.7554/eLife.81083

Figure Lengend Snippet: ( A ) MR49F cells were treated with GCN2iB (2 µM) or vehicle control (DMSO) for 96 hr in normal growth media, growth media supplemented with essential amino acid (EAA), or growth media supplemented with individual amino acids as indicated. Cell growth was quantified using CellTiter-Glo as described in the Materials and methods and is presented normalized to the vehicle control group. ( B ) 22Rv1 WT and 22Rv1 GCN2 KO cells were cultured for 96 hr in normal growth media, growth media supplemented with EAA, or growth media supplemented with individual amino acids as indicated and cell growth was similarly measured as described in A . Statistical significance in panels A and B was determined using a one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons. Error bars indicate standard deviation (SD) ( N = 3); ****p ≤ 0.0001.

Article Snippet: Media was changed to standard culture media containing 500 nM to 10 µM GCN2iB or DMSO as a control or standard media lacking histidine (MyBiosource, Cat. #MBS652918) and incubated for the indicated time period.

Techniques: Cell Culture, Standard Deviation

Journal: eLife

Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

doi: 10.7554/eLife.81083

Figure Lengend Snippet: ( A ) Gene-level depletion for LNCaP and 22Rv1 cells. The average log2 fold change for the single guide RNAs (sgRNAs) for each gene is shown on the x -axis. Significantly depleted genes (p ≤ 0.05) in LNCaP or 22Rv1 are indicated. Circle size indicates the number of significant sgRNAs. SLC genes in red are dependent on GCN2 for expression. ( B ) Plot of −Log 10 (p value) for depleted genes identified in CRISPR screen for LNCaP versus 22Rv1 cells. Significantly depleted genes (p ≤ 0.05) in LNCaP, 22Rv1 or both cell lines are indicated. SLC genes in red are GCN2 dependent. ( C ) Lysates from LNCaP cells were treated with 2 µM GCN2iB for 6 or 24 hr, or with vehicle (DMSO) were analyzed by immunoblot analyses using antibodies that recognize total or phosphorylated GCN2-T899, ATF4, 4F2 (SLC3A2), or actin. Molecular weight markers are indicated in kilodaltons for the panels. ( D ) LNCaP cells were cultured in standard culture conditions (NM: normal media), media supplemented with 200 µM histidine (+His), or media depleted of histidine (−His) for 24 hr. Lysates were analyzed by immunoblot analyses using antibodies that recognize total or phosphorylated GCN2-T899, ATF4, 4F2 (SLC3A2), or actin. ( E ) LNCaP cells were treated with 100 nM halofuginone (HF) for 2 and 6 hr or vehicle (DMSO). Lysates were analyzed by Immunoblot using antibodies that recognize the indicated proteins. ( F ) 4F2 (SLC3A2) expression was reduced in LNCaP or 22Rv1 cells using two different siRNAs or scramble siRNA as a control. Cell growth was measured in replicate wells ( N = 5) for up to 6 days and are plotted relative to day 0 (mean ± standard deviation [SD]). Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001. ( G ) LNCaP cells transfected with two different siRNAs targeting 4F2 (SLC3A2) or scramble siRNA for 48 hr. Lysate was prepared and analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2-T899, total or phosphorylated eIF2α−S51, ATF4, 4F2 (SLC3A2), or actin. ( H ) LNCaP cells stably overexpressing SLC3CA2 or vector control were transfected with two different siRNAs targeting GCN2 or scrambled control. Cells were then treated with GCN2iB (2 µM) or vehicle and growth was measured in replicate wells ( N = 5) and is plotted relative to day 0 (mean ± SD). Statistical significance was determined using a two-way ANOVA as described in ; **p ≤ 0.01, ****p ≤ 0.0001. ( I ) Amino acid measurements of LNCaP cells transfected siRNA targeting GCN2 ( N = 4), 4F2 (SLC3A2, N = 4), or scramble control ( N = 8). Two separate bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined using a two-way ANOVA as described in . Error bars indicate SD; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.

Article Snippet: Media was changed to standard culture media containing 500 nM to 10 µM GCN2iB or DMSO as a control or standard media lacking histidine (MyBiosource, Cat. #MBS652918) and incubated for the indicated time period.

Techniques: Expressing, CRISPR, Western Blot, Molecular Weight, Cell Culture, Standard Deviation, Transfection, Stable Transfection, Plasmid Preparation

Journal: eLife

Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

doi: 10.7554/eLife.81083

Figure Lengend Snippet: ( A ) 4F2 (SLC3A2) and ATF4 mRNA were measured by qRT-PCR as described in the Materials and methods in LNCaP cells treated with 2 µM GCN2iB for 6 or 24 hr or vehicle control (DMSO), ( B ) cultured in standard culture conditions (NM: normal media), media supplemented with 200 µM histidine (+His), or media depleted of histidine (− His) for 24 hr, or ( C ) treated with 100 nM halofuginone (HF) for 2 or 6 hr or untreated (DMSO control). Error bars indicate standard deviation (SD) ( N = 3). An unpaired two-tailed t -test was used to determine statistical significance; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Article Snippet: Media was changed to standard culture media containing 500 nM to 10 µM GCN2iB or DMSO as a control or standard media lacking histidine (MyBiosource, Cat. #MBS652918) and incubated for the indicated time period.

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

Journal: eLife

Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

doi: 10.7554/eLife.81083

Figure Lengend Snippet: ( A ) LNCaP cells were treated with GCN2iB (2 µM) or vehicle (DMSO) control in the presence or absence of salubrinal (50 µM) for 48 hr. Protein lysates were prepared and analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2, total or phosphorylated eIF2α, ATF4, 4F2 (SLC3A2), or actin as indicated. ( B ) LNCaP cells transfected with empty vector (EV) control or pMSCV-GADD34-puro expression plasmid encoding the human GADD34 gene were analyzed by immunoblot as indicated in panel A. ( C ) Protein lysates prepared from LNCaP or 22Rv1 stably expressing empty vector (EV) control or 4F2 (SLC3A2) were analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2, total or phosphorylated eIF2α(S-51), ATF4, or actin as indicated. ( D ) Growth of LNCaP and 22Rv1 cells stably expressing empty vector (EV) control or 4F2 (SLC3A2) was measured in replicate wells ( N = 5) for up to 4 days and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001.

Article Snippet: Media was changed to standard culture media containing 500 nM to 10 µM GCN2iB or DMSO as a control or standard media lacking histidine (MyBiosource, Cat. #MBS652918) and incubated for the indicated time period.

Techniques: Western Blot, Transfection, Plasmid Preparation, Expressing, Stable Transfection, Standard Deviation

Journal: eLife

Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

doi: 10.7554/eLife.81083

Figure Lengend Snippet: Male NSG mice were injected subcutaneously with LNCaP ( N = 5) ( A ) or 22Rv1 ( N = 4) ( B ) cells, or alternatively implanted with tumor fragments from an androgen-sensitive tumor TM00298 ( N = 5) ( C ). Male castrated NSG mice were implanted with tumor fragments from LuCaP-35 CR tumors ( N = 5) ( D ). Mice were treated with vehicle or 30 mg/kg GCN2iB twice daily for 5 days/week and tumor volumes were measured on indicated days. Statistical significance was determined using a two-way analysis of variance (ANOVA) with Sidak’s multiple comparison. Error bars indicate standard error of the mean (SEM); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Final tumor weight was measured at endpoint and is represented in bar graphs (right panels). Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD); *p ≤ 0.05. ( E ) Protein lysates were prepared from 22Rv1 tumors treated with vehicle or GCN2iB and analyzed by immunoblot for phosphorylated GCN2-T899, ATF4, LAT1 (SLC7A5), xCT (SLC7A11), and 4F2 (SLC3A2), and actin. The levels of the SLC proteins normalized to actin are shown. Phosphorylated GCN2-T899 was normalized to total GCN2. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 4); *p ≤ 0.05. ( F ) Amino acid measurements of 22Rv1 tumors treated with vehicle or GCN2iB. Bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 4); # p ≤ 0.1; *p ≤ 0.05; **p ≤ 0.01. ( G ) Pearson correlation between p-GCN2-T899 and 4F2 (SLC3A2) histoscores calculated from IHC staining from a prostate tumor microarray (Biomax PR807c) containing normal ( N = 10), hyperplasia ( N = 20), and malignant ( N = 50) for all tissues (combined) or Gleason scores 4 and 5. The center lines depict linear regression (95% confidence intervals). Not all samples were analyzed due to damaged/quality of tissue samples. Levels of p-GCN2-T899 and 4F2 (SLC3A2) were measured by IHC staining and QuPath was used to determine the histoscore. Two representative cases are shown for high (Case 1) and low (Case 2) p-GCN2-T899 and 4F2 (SLC3A2) staining. Scale bar indicates 200 µm (main image) and 20 µm (insert). ( H ) Correlation of expression of 4F2 (SLC3A2) and a GCN2-dependent gene signature in prostate adenocarcinoma (PRAD, N = 551) from the Cancer Genome Atlas (TCGA). The GCN2-dependepent gene signature was derived from RNA-seq data as described in the Materials and methods.

Article Snippet: Media was changed to standard culture media containing 500 nM to 10 µM GCN2iB or DMSO as a control or standard media lacking histidine (MyBiosource, Cat. #MBS652918) and incubated for the indicated time period.

Techniques: Injection, Two Tailed Test, Standard Deviation, Western Blot, Immunohistochemistry, Microarray, Staining, Expressing, Derivative Assay, RNA Sequencing Assay

Journal: eLife

Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

doi: 10.7554/eLife.81083

Figure Lengend Snippet: Male NSG mice were injected subcutaneously with LNCaP ( N = 5) or 22Rv1 ( N = 4) cells, or implanted with tumor fragments from an androgen-sensitive tumor TM00298 ( N = 5) or the castration-resistant LuCaP-35 CR ( N = 5) tumor and treated as described in . ( A ) Representative images showing IHC staining for Ki-67 or p-GCN2-T899 from tumor described above. Bar graphs show quantification of percent positive nuclear staining (Ki67) or Histoscore (p-GCN2-T899). Error bars indicate standard deviation (SD). Statistical significance was determined using an unpaired two-tailed t -test; *p ≤0.05; **p ≤ 0.01. ( B ) Mouse body weight was measured on indicated days for mice bearing LNCaP, 22Rv1, TM00298, or LuCaP-35 CR tumors treated with vehicle or GCN2iB as described in .

Article Snippet: Media was changed to standard culture media containing 500 nM to 10 µM GCN2iB or DMSO as a control or standard media lacking histidine (MyBiosource, Cat. #MBS652918) and incubated for the indicated time period.

Techniques: Injection, Immunohistochemistry, Staining, Standard Deviation, Two Tailed Test

Journal: eLife

Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

doi: 10.7554/eLife.81083

Figure Lengend Snippet: Immunoblot analysis of 22Rv1 ( A ) or TM00298 ( B ) tumors from mice treated with GCN2iB as described in . Molecular weight markers are indicated in kilodaltons for each immunoblot panel. The levels of the indicated proteins from TM00298 tumors normalized to appropriate control are shown in the bar graph ( N = 4).

Article Snippet: Media was changed to standard culture media containing 500 nM to 10 µM GCN2iB or DMSO as a control or standard media lacking histidine (MyBiosource, Cat. #MBS652918) and incubated for the indicated time period.

Techniques: Western Blot, Molecular Weight