Journal: Theranostics

Article Title: A natural small molecule isoginkgetin alleviates hypercholesterolemia and atherosclerosis by targeting ACLY

doi: 10.7150/thno.105782

Figure Lengend Snippet: Discovery of Isoginkgetin as a naturally-occurring ACLY inhibitor. (A) Schematic diagram of molecular autodocking. Briefly, we conducted virtual molecular docking based on the structure of the ACLY protein to screen for potential natural small molecules that interact with ACLY from a natural product library comprising 3,200 compounds. (B) The predicted affinity results of A. (C) The inhibitory effect of the top candidates on ACLY enzyme activity was evaluated in vitro using the ADP-Glo assay (n = 3). One-way ANOVA followed by Bonferroni's post hoc test. Compared with compound 0 µM. (D) Chemical structure of ISOGK. (E) The cell viability in primary hepatocytes from C57BL/6J mouse treated with dose-dependent ISOGK detected by CCK-8 assay (n = 4). One-way ANOVA followed by Bonferroni's post hoc test. Compared with ISOGK 0 µM. (F) The cell viability in HepG2 cell line treated with dose-dependent ISOGK detected by CCK-8 assay (n = 3, technical replicates). One-way ANOVA followed by Bonferroni's post hoc test. Compared with ISOGK 0 µM. (G) Representative images of Nile Red staining in primary hepatocytes isolated from mice fed a high fat diet for 16 weeks which are treated with vehicle or indicated concentrations ISOGK for 12 h. (H) Quantitative analysis of G (n = 3). One-way ANOVA followed by Bonferroni's post hoc test. The data are means ± SEM, n.s., not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: C57BL/6J mice had a standard chow diet for 4 weeks with concurrent treatment with ISOGK (HY-N2117, MedChemExpress, Piscataway, NJ, USA).

Techniques: Activity Assay, In Vitro, Glo Assay, CCK-8 Assay, Staining, Isolation

Journal: Theranostics

Article Title: A natural small molecule isoginkgetin alleviates hypercholesterolemia and atherosclerosis by targeting ACLY

doi: 10.7150/thno.105782

Figure Lengend Snippet: Isoginkgetin attenuates atherosclerosis in male and female Apoe -/- mice. (A) Scheme of study design. Male and female Apoe -/- mice were induced by high cholesterol diet for 7 weeks. Animals then received ISOGK treatment (20 mg/kg/day) for another 8 weeks. (B) The body weights were shown after ISOGK treatment for 8 weeks (n = 10 or 11). Two-tailed Student's t test. (C) Serum ALT, AST levels of Apoe -/- mice treated with or without ISOGK (n = 10 or 11). Two-tailed Student's t test. (D) Representative images of Oil Red O staining of en face aortas from male and female Apoe -/- mice fed high cholesterol diet. (E) Quantification of en face plaque areas as a percentage of the area in D (n = 10 or 11). Two-tailed Student's t test. (F) The representative images of Oil Red O (upper) staining and H&E (lower) staining of the aortic sinus from male and female Apoe -/- mice fed high cholesterol diet. Arrow indicates necrotic core area. (G) Quantitative analysis of F (n = 6 or 7). Two-tailed Student's t test. The data are means ± SEM, n.s., not significant, ** P < 0.01, *** P < 0.001.

Article Snippet: C57BL/6J mice had a standard chow diet for 4 weeks with concurrent treatment with ISOGK (HY-N2117, MedChemExpress, Piscataway, NJ, USA).

Techniques: Two Tailed Test, Staining

Journal: Theranostics

Article Title: A natural small molecule isoginkgetin alleviates hypercholesterolemia and atherosclerosis by targeting ACLY

doi: 10.7150/thno.105782

Figure Lengend Snippet: Isoginkgetin modulates lipid metabolism in Apoe -/- mouse liver. (A) Scheme of sample processing. (B) Volcano plot showing differential expression genes (adjusted P < 0.05, | log2 (Fold change) | > 1). (C) Heat map displaying the indicated gene expression in liver tissues from Apoe -/- mice with or without ISOGK (n = 4). (D) GO enrichment analysis of transcriptomes from Apoe -/- mice liver samples. (E) KEGG pathway enrichment analysis of transcriptomes from Apoe -/- mice liver samples. (F) GSEA analysis of transcriptomes from Apoe -/- mice liver samples. The data are means ± SEM, n.s., not significant, * P < 0.05, ** P < 0.01, **** P < 0.0001.

Article Snippet: C57BL/6J mice had a standard chow diet for 4 weeks with concurrent treatment with ISOGK (HY-N2117, MedChemExpress, Piscataway, NJ, USA).

Techniques: Expressing, Gene Expression

Journal: Theranostics

Article Title: A natural small molecule isoginkgetin alleviates hypercholesterolemia and atherosclerosis by targeting ACLY

doi: 10.7150/thno.105782

Figure Lengend Snippet: Isoginkgetin displays lipid-lowering effects in hyperlipidemia mice and hamsters. (A) Serum levels of TC, TG, LDL-C, HDL-C, and hepatic levels of TC, TG from male and female Apoe -/- mice fed high cholesterol diet 7 weeks and administrated ISOGK 8 weeks (n = 10 or 11). Two-tailed Student's t test. (B) The distribution of TC and TG in pooled plasma samples from the indicated female Apoe -/- mice. (C) Study design of the hamster's experiment. (D) Plasma levels of TC, TG, LDL-C, HDL-C, and hepatic levels of TC, TG in the indicated hamster's group after treatment ISOGK or vehicle for 4 weeks (n = 8 - 10). One-way ANOVA followed by Bonferroni's post hoc test. (E) The distribution of TC and TG in pooled plasma samples from the indicated hamsters. (F) Representative images of Oil Red O staining in liver sections from the indicated groups. (G) Quantitative analysis of F (n = 8). One-way ANOVA followed by Bonferroni's post hoc test. The data are means ± SEM, n.s., not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: C57BL/6J mice had a standard chow diet for 4 weeks with concurrent treatment with ISOGK (HY-N2117, MedChemExpress, Piscataway, NJ, USA).

Techniques: Two Tailed Test, Staining

Journal: Theranostics

Article Title: A natural small molecule isoginkgetin alleviates hypercholesterolemia and atherosclerosis by targeting ACLY

doi: 10.7150/thno.105782

Figure Lengend Snippet: Isoginkgetin ameliorates hyperlipidemia and atherosclerosis in Ldlr -/- hamsters. (A) Scheme of study design. Briefly, 8-week-old male Ldlr -/- hamsters were induced by high cholesterol diet for 2 weeks and then administrated ISOGK (2 mg/kg/day and 5 mg/kg/day) treatment for another 6 weeks. (B) Hepatic levels of TC, TG in the indicated Ldlr -/- hamster's group after treatment ISOGK or vehicle for 8 weeks (n = 6 or 7). One-way ANOVA followed by Bonferroni's post hoc test. (C) Serum TC, TG, HDL-C and non-HDL-C levels in the indicated Ldlr -/- hamster's group after treatment ISOGK or vehicle for 6 weeks (n = 7). One-way ANOVA followed by Bonferroni's post hoc test. (D) Representative images of Oil Red O staining of en face aortas in indicated Ldlr -/- hamsters group. (E) Quantitative analysis of D (n = 6 or 7). One-way ANOVA followed by Bonferroni's post hoc test. (F) Representative images of Oil Red O staining in Ldlr -/- hamsters' aortic sinus and representative images of Nile Red staining in Ldlr -/- hamsters' liver tissues. (G-H) Quantitative analysis of F (n = 6 or 7). One-way ANOVA followed by Bonferroni's post hoc test. The data are means ± SEM, n.s., not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: C57BL/6J mice had a standard chow diet for 4 weeks with concurrent treatment with ISOGK (HY-N2117, MedChemExpress, Piscataway, NJ, USA).

Techniques: Staining

Journal: Theranostics

Article Title: A natural small molecule isoginkgetin alleviates hypercholesterolemia and atherosclerosis by targeting ACLY

doi: 10.7150/thno.105782

Figure Lengend Snippet: Isoginkgetin inhibits ACLY activity in vitro and in vivo. (A) Primary hepatocytes from C57BL/6J mice were cultured with vehicle or 5 µM ISOGK for 4 h in the presence of [1,2- 14 C]-acetate. The hepatic lipogenesis was calculated (n = 3). One-way ANOVA followed by Bonferroni's post hoc test. (B) Dose-dependent inhibition of purified, recombinant ACLY after incubation with ISOGK, the activity at control was defined as 100%. (C) ACLY enzyme activities were measured from the liver extracts of male and female Apoe -/- mice fed high cholesterol diet 7 weeks and administrated ISOGK 8 weeks (n = 10 or 11). Two-tailed Student's t test. (D). ACLY enzyme activities were measured from the liver extracts of hamster after treatment ISOGK or vehicle for 4 weeks (n = 10). One-way ANOVA followed by Bonferroni's post hoc test. (E) ACLY enzyme activities were measured from the liver extracts of Ldlr -/- hamster after treatment with or without ISOGK for 8 weeks (n = 6 or 7). One-way ANOVA followed by Bonferroni's post hoc test. (F) Molecular docking has predicted the binding sites of ISOGK with ACLY. (G) The interaction of ACLY with ISOGK was measured by surface plasmon resonance. (H) CETSA analyzed the thermal stabilization of ACLY with isoginkgetin in AML12 cell lysates. (I) Quantitative analysis of H (n = 3). Two-way ANOVA with Dunnett's T3 post hoc analysis. The data are means ± SEM, n.s., not significant, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: C57BL/6J mice had a standard chow diet for 4 weeks with concurrent treatment with ISOGK (HY-N2117, MedChemExpress, Piscataway, NJ, USA).

Techniques: Activity Assay, In Vitro, In Vivo, Cell Culture, Inhibition, Purification, Recombinant, Incubation, Control, Two Tailed Test, Binding Assay, SPR Assay

Journal: Theranostics

Article Title: A natural small molecule isoginkgetin alleviates hypercholesterolemia and atherosclerosis by targeting ACLY

doi: 10.7150/thno.105782

Figure Lengend Snippet: The anti-atherosclerotic and lipid-lowering effects of isoginkgetin in Apoe -/- mice were ACLY-dependent. (A) Scheme of study design. Briefly, male Apoe -/- mice were induced by high cholesterol diet for 7 weeks and administrated GalNAc-siAcly (3 mg/kg/month). Animals then received ISOGK (20 mg/kg/day) treatment for another 8 weeks. (B) Immunoblotting analysis and quantification of ACLY protein in liver and kidney tissues from indicated groups (n = 3). One-way ANOVA followed by Bonferroni's post hoc test. (C) Representative images of Oil Red O staining of en face aortas from male Apoe -/- mice fed high cholesterol diet. (D) Representative images of ORO staining of the aortic sinus from male Apoe -/- mice fed high cholesterol diet. (E) Representative images of H&E staining of the aortic sinus from male Apoe -/- mice fed High cholesterol diet. (F) Quantitative analysis of C-E (n = 6 - 8). One-way ANOVA followed by Bonferroni's post hoc test. (G) Plasma levels of TC, TG, LDL-C and HDL-C in the indicated groups (n = 6 - 8). One-way ANOVA followed by Bonferroni's post hoc test. The data are means ± SEM, n.s., not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: C57BL/6J mice had a standard chow diet for 4 weeks with concurrent treatment with ISOGK (HY-N2117, MedChemExpress, Piscataway, NJ, USA).

Techniques: Western Blot, Staining

Journal: PLOS ONE

Article Title: Isoginkgetin and Madrasin are poor splicing inhibitors

doi: 10.1371/journal.pone.0310519

Figure Lengend Snippet: (A) Chemical structure of Madrasin and Isoginkgetin. (B) RT-PCR with primers amplifying the intron located between exons 2 and 3 of DNAJB1 or exons 4 and 5 of BRD2 . HeLa cells were treated with DMSO, 1 μM PlaB, 1 μM HB, 30 μM Isoginkgetin, or 90 μM Madrasin for 1h. The location of the spliced and unspliced RNA is shown on the left of the panel. The percentages of unspliced RNA compared to total (spliced + unspliced) are shown below. (C) Representative images of immunofluorescence analysis or 5’EU incorporation in HeLa cells treated with DMSO, 100 μM DRB, a CDK9 inhibitor, 1 μM PlaB, 1 μM HB, 30 μM Isoginkgetin, or 90 μM Madrasin for 1h. EU (green), DAPI (blue), scale bars: 50 μm. (D) Quantification of 5’EU intensity per nucleus for DMSO, DRB, PlaB, HB, Isoginkgetin, and Madrasin. Boxplot settings are: min to max values with the box showing 25–75 percentile range. > 10,000 nuclei were quantified per condition. Statistical test: Kruskal-Wallis test. P-value: n.s. not significant, **** < 0.0001. ( E ) Western blots of total pol II, SF3B1, α-tubulin (loading control), and histone H3 (loading control) from chromatin, nucleoplasm, and cytoplasm fractions of HeLa cells treated with DMSO, 1 μM PlaB, 1 μM HB, 30 μM Isoginkgetin, or 90 μM Madrasin for 1h.

Article Snippet: HeLa cells were treated with 30–90 μM Madrasin (Sigma-Aldrich), 30 μM Isoginkgetin (Merck), 1 μM Herboxidiene (Cayman Chemical), 1 μM Pladienolide B (Cambridge Bioscience), or 100 μM 5,6-dichlorobenzimidazone-1-β-D-ribofuranoside (DRB, Sigma) for the time indicated in the figures.

Techniques: Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Western Blot, Control

Journal: PLOS ONE

Article Title: Isoginkgetin and Madrasin are poor splicing inhibitors

doi: 10.1371/journal.pone.0310519

Figure Lengend Snippet: (A) RT-PCR with primers amplifying the intron located between exons 2 and 3 of DNAJB1 or the intron located between exons 4 and 5 of BRD2 . HeLa cells were treated with DMSO, 30 μM Isoginkgetin for 1h to 6h, or 1 μM PlaB for 6h. The locations of the spliced and unspliced RNA are shown on the right of the panel. The percentages of unspliced RNA compared to total (spliced + unspliced) are shown below. (B) Percentage of spliced reads in total RNA-seq treated with DMSO (blue) or Isoginkgetin (red) for the time indicated on the figure. Each point represents a biological replicate. Statistical test: Wilcoxon rank sum test. P-value: n.s. not significant, * < 0.05, **** < 0.0001. (C) Representative images of immunofluorescence analysis or 5’EU incorporation in HeLa cells treated with DMSO or 30 μM Isoginkgetin for 1h to 6h. EU (green), DAPI (blue), scale bars: 50 μm. (D) Quantification of 5’EU intensity per nucleus for DMSO and Isoginkgetin. Boxplot settings are: min to max values with the box showing 25–75 percentile range. 8,466 nuclei were quantified per condition. Statistical test: Kruskal-Wallis test. P-value: **** < 0.0001. (E) qRT-PCR with primers amplifying a region from the pol I transcribed pre-rRNA. HeLa cells were treated with DMSO or 30 μM Isoginkgetin for 1h to 6h. cDNA was generated with random hexamers. Values are normalised to the 7SK snRNA and shown as relative to DMSO, mean ± SEM, n = 3 biological replicates. Statistical test: two-tailed unpaired t-test. P-value: n* < 0.05.

Article Snippet: HeLa cells were treated with 30–90 μM Madrasin (Sigma-Aldrich), 30 μM Isoginkgetin (Merck), 1 μM Herboxidiene (Cayman Chemical), 1 μM Pladienolide B (Cambridge Bioscience), or 100 μM 5,6-dichlorobenzimidazone-1-β-D-ribofuranoside (DRB, Sigma) for the time indicated in the figures.

Techniques: Reverse Transcription Polymerase Chain Reaction, RNA Sequencing Assay, Immunofluorescence, Quantitative RT-PCR, Generated, Two Tailed Test

Journal: PLOS ONE

Article Title: Isoginkgetin and Madrasin are poor splicing inhibitors

doi: 10.1371/journal.pone.0310519

Figure Lengend Snippet: (A) qRT-PCR with primers amplifying different protein-coding genes. HeLa cells were treated with DMSO or 30 μM Isoginkgetin for 1h to 6h. cDNA was generated with oligo(dT). Values are normalised to the protein-coding gene GAPDH and shown as relative to DMSO, mean ± SEM, n = 4 biological replicates. Statistical test: two-tailed unpaired t-test. P-value: * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001. (B) Total pol II ChIP-qPCR on the intron-containing gene KPNB1 in HeLa cells treated with DMSO or 30 μM Isoginkgetin for 1h to 6h. Mean ± SEM, n = 3 biological replicates. Statistical test: two-tailed unpaired t-test. P-value: * < 0.05, ** < 0.01. (C) Total pol II ChIP-qPCR on the intronless gene JUN in HeLa cells treated with DMSO or 30 μM Isoginkgetin for 1h to 6h. Mean ± SEM, n = 3 biological replicates. Statistical test: two-tailed unpaired t-test. P-value: * < 0.05. (D) Total pol II ChIP-qPCR of the histone gene H1-2 in HeLa cells treated with DMSO or 30 μM Isoginkgetin for 1h to 6h. Mean ± SEM, n = 3 biological replicates. Statistical test: two-tailed unpaired t-test. P-value: * < 0.05. (E) Western blots of total pol II, Ser2-P, Ser5-P, and histone H3 on the chromatin fraction of HeLa cells treated with DMSO or 30 μM Isoginkgetin for 1h, 2h, or 4h. Quantifications of the western blots are shown below each panel. Each quantification has been normalised to histone H3 signal and then to the DMSO condition. (F) ChIP-qPCR of total pol II, Ser2-P, and Ser5-P across the intron-containing gene KPNB1 in HeLa cells treated with DMSO or 30 μM Isoginkgetin for 1h, 1h30, or 2h. Mean ± SEM, n = 3 biological replicates. Statistical test: two-tailed unpaired t-test. P-value: * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001.

Article Snippet: HeLa cells were treated with 30–90 μM Madrasin (Sigma-Aldrich), 30 μM Isoginkgetin (Merck), 1 μM Herboxidiene (Cayman Chemical), 1 μM Pladienolide B (Cambridge Bioscience), or 100 μM 5,6-dichlorobenzimidazone-1-β-D-ribofuranoside (DRB, Sigma) for the time indicated in the figures.

Techniques: Quantitative RT-PCR, Generated, Two Tailed Test, Western Blot

Journal: Nucleic Acids Research

Article Title: Nuclear RNA-related processes modulate the assembly of cytoplasmic RNA granules

doi: 10.1093/nar/gkae119

Figure Lengend Snippet: Splicing inhibition by isoginkgetin prevents the formation of arsenite-induced SGs. ( A ) U2OS cells were treated with isoginkgetin (Iso, 100 μM; 4 h) and arsenite (0.25 mM) was added 30 min before fixation. Cells were stained with anti-G3BP1 as an SG marker. Scale bar = 10 μm. ( B ) Quantification of the population of SG-positive U2OS cells treated as described in panel (A). Cells were counted in three independent experiments ( n > 180 cells per treatment). Data were analyzed with independent sample t -tests (*** P < 0.001). Bar graph illustrates the mean ± standard deviation. ( C ) Frames from live-cell movies of GFP-IGF2B3 stably expressing cells under the following conditions: untreated, treated with arsenite, treated with isoginkgetin (100 μM; 4 h) and followed by the addition of arsenite (0.25 mM) at the starting time point ( t = 0 min). Scale bar = 10 μm. ( D ) Western blot of protein extracts from U2OS cells treated with arsenite (Ars, 0.25 mM, 1 h), isoginkgetin (100 μM, 4 h), PLB (0.5 μM, 6 and 24 h) and madrasin (30 μM, 4 h). Blots were incubated with anti-eIF2α, anti-p-eIF2α and anti-tubulin for loading control. ( E ) Quantification of the eIF2α phosphorylation levels from panel (D). Phosphorylation levels in all samples were normalized to the total eIF2α protein level in each sample, and all samples were normalized to the untreated signal, and then the data were log transformed. Data were analyzed using ImageJ, and statistical analysis was carried out by one-way ANOVA followed by Tukey’s post hoc analysis ( n = 3, * P < 0.05, ** P < 0.01, *** P < 0.001). Bar graph illustrates the mean ± standard deviation.

Article Snippet: For splicing inhibition, cells were treated with pladienolide B (PLB; 0.5 μM, Santa Cruz, 445493) for 6 or 24 h, with isoginkgetin (50–100 μM, EMD Millipore, purchased from Sigma, 416154) for 4 h or with madrasin (20–30 μM, Sigma, SML1409) for 4 h, all dissolved in dimethyl sulfoxide (DMSO) (Sigma, D2650).

Techniques: Inhibition, Staining, Marker, Standard Deviation, Stable Transfection, Expressing, Western Blot, Incubation, Transformation Assay

Journal: Nucleic Acids Research

Article Title: Nuclear RNA-related processes modulate the assembly of cytoplasmic RNA granules

doi: 10.1093/nar/gkae119

Figure Lengend Snippet: The effect of splicing inhibitors on PBs. ( A ) U2OS cells were treated with PLB (0.5 μM) for 6 or 24 h, and cells were stained with anti-Dcp1a to mark PBs (green). DNA Hoechst staining is in cyan. Scale bars = 10 μm. ( B ) Quantifications representing average PB numbers per cell. U2OS cells were treated as described in panel (A), and then data were log transformed (base 2) to meet normality assumption. Data were analyzed using ANOVA, followed by Tukey’s post hoc analysis ( n = 3, >220 cells were quantified per treatment). Bar graph illustrates mean ± standard deviation. ( C ) Violin plot representing PB area in U2OS cells treated as described in panel (A). Data were analyzed using one-way nested ANOVA. More than 330 PBs were quantified for each experiment per treatment ( n = 3). The graph represents one experiment, and the statistical test for significance was carried for all three independent experiments (*** P < 0.001, * P < 0.05). ( D ) U2OS cells were treated with madrasin (30 μM; 4 h). Arsenite (0.25 mM) was added 30 min before fixation, and cells were stained with anti-Dcp1a to mark PBs (green) and anti-G3BP1 to mark SGs (magenta). ( E ) U2OS cells were treated with isoginkgetin (100 μM; 4 h). Arsenite (0.25 mM) was added 30 min before fixation, and cells were stained with anti-Dcp1a to mark PBs (green) and anti-G3BP1 to mark SGs (magenta). ( F ) Quantifications representing average PB numbers per cell. U2OS cells were treated as described in panel (D). Data were analyzed as described in panel (B), and >200 cells were quantified per treatment ( n = 3, *** P < 0.001, ns = nonsignificant). ( G ) Quantifications representing average PB numbers per cell. U2OS cells were treated as described in panel (E). Data were analyzed as described in panel (B), and >290 cells were quantified per treatment ( n = 3, *** P < 0.001, ns = nonsignificant).

Article Snippet: For splicing inhibition, cells were treated with pladienolide B (PLB; 0.5 μM, Santa Cruz, 445493) for 6 or 24 h, with isoginkgetin (50–100 μM, EMD Millipore, purchased from Sigma, 416154) for 4 h or with madrasin (20–30 μM, Sigma, SML1409) for 4 h, all dissolved in dimethyl sulfoxide (DMSO) (Sigma, D2650).

Techniques: Staining, Transformation Assay, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Nuclear RNA-related processes modulate the assembly of cytoplasmic RNA granules

doi: 10.1093/nar/gkae119

Figure Lengend Snippet: mRNA transfection partially rescues SG formation under splicing inhibition followed by arsenite treatment. ( A ) U2OS cells were treated with isoginkgetin (Iso, 50 μM) for 5 h overall, transfected with mRNA (0.75–1 μg) encoding GFP (green) for 1.5 h and treated with arsenite 30 min before fixation. Cells were stained with anti-G3BP1 to mark SGs (cyan). Scale bar = 10 μm. ( B ) Quantification of the population of SG-positive U2OS cells treated as described in panel (A). Cells were counted in three independent experiments ( n > 260 cells per treatment). Data were analyzed with independent sample t -tests or with one-way ANOVA, followed by Tukey’s post hoc analysis (*** P < 0.001). Bar graph illustrates the mean ± standard deviation. ( C ) Illustration of the suggested model. (Left) Under stress conditions, SGs are formed. (Middle) Shortage of mRNA in the cytoplasm due to the inhibition of splicing or export prevents the formation of SGs under stress conditions. (Right) Excess of cytoplasmic mRNA leads to SG formation, via the activation of PERK and GCN2, without applying any additional stress.

Article Snippet: For splicing inhibition, cells were treated with pladienolide B (PLB; 0.5 μM, Santa Cruz, 445493) for 6 or 24 h, with isoginkgetin (50–100 μM, EMD Millipore, purchased from Sigma, 416154) for 4 h or with madrasin (20–30 μM, Sigma, SML1409) for 4 h, all dissolved in dimethyl sulfoxide (DMSO) (Sigma, D2650).

Techniques: Transfection, Inhibition, Staining, Standard Deviation, Activation Assay