Journal: Science Advances

Article Title: Stress granule dynamics govern TOR reactivation and growth recovery during post-heat stress adaptation

doi: 10.1126/sciadv.adv6202

Figure Lengend Snippet: ( A ) TOR activity was strongly inhibited by Torin2, AZD8055, and glucose depletion in 4-day-old 35Spro::S6K1-HA/Col-0 seedlings. TOR activity was assessed by Western blot using an antibody against phosphorylated Thr 449 in S6K1 (S6K1-pT449). Glc, glucose. Torin2 and AZD8055, TOR kinase inhibitors, which were pretreated for 1 hour before sample collection. ( B ) Quantification data of (A). Three biological repeats. Mean ± SD. Statistical significance was determined by one-way analysis of variance (ANOVA). * indicates P value < 0.05. ( C ) Inhibition of TOR did not trigger SG formation in the roots of PAB4pro::PAB4-GFP and RBP47bpro::GFP-RBP47b seedlings. Scale bars, 10 μm. Torin2 and AZD8055 were pretreated for 1 hour before imaging. ( D ) Heat-induced SG formation was independent of TOR signaling. HS, heat stress treatment. Scale bars, 10 μm. Torin2 and AZD8055 were pretreated for 1 hour before heat treatment.

Article Snippet: Torin2 (1 μM, Tocris Bioscience, catalog no. 4248), AZD8055 (1 μM, Selleckchem, catalog no. S1555), and CHX (200 ng/μl; INALCO, catalog no. 1758-9310) were dissolved in dimethyl sulfoxide (DMSO).

Techniques: Activity Assay, Western Blot, Inhibition, Imaging

Journal: bioRxiv

Article Title: Glucose metabolism distinguishes TE from ICM fate during mammalian embryogenesis

doi: 10.1101/826875

Figure Lengend Snippet: ( A-D ) AP-2γ translation is controlled by PPP function. Phosphorylation of threonine 37/46 on 4E-BP1 (p-4E-BP1), a target of mTOR is expressed at high levels in the 8-cell embryo ( A ). This expression is lost in –G ( B ) or when PPP is inhibited ( C ). Quantitation in ( D ) also shows that blocking HBP does not affect p-4E-BP1. ( E, F ) Western blot analysis of members of mTOR signaling pathway and its activation. ( E ) Western blot analysis shows that total mTOR, TSC2, phosphorylation level of serine 473 on AKT1/2 (p-AKT) and serine 127 on YAP1 (p-YAP1) all remain unchanged in embryos grown without glucose when compared to +G control. In contrast, phosphorylated mTOR targets 4E-BP1 and RPS6 (p-RPS6) levels are significantly reduced in –G embryos. ( F ) p-RPS6 is eliminated when S1PR 2 (JTE), Rac1 (NSC) or mTOR (INK) is inhibited. ( G-J ) AP-2γ expression requires mTOR and S1P signaling. The expression of AP-2γ ( G ) is eliminated in the presence of mTOR inhibitors, Torin2 and INK ( H, I ) and S1PR 2 inhibitor JTE ( J ). An identical effect is seen on CDX2 expression . ( K ) Quantitation of AP-2γ expression in embryos in which the mTOR and S1P pathways are inhibited. ( L ) Quantitation shows that the morula to blastocyst transition is acutely sensitive to mTOR inhibitors (Torin2, INK128), but is not affected by any of the seven PI3K inhibitors tested (BLY719, BKM120, GDC0941, TGX221, BX912, GSK2334470, MK2201). However, blastocyst formation is extremely sensitive to inhibitors of the S1P pathway (JTE013, SKI-II) and its downstream component Rac1 (NSC23766). ( M-T ) Role of glucose and S1P signaling in controlling Rac1 expression. In control, Rac1-GTP staining is observed only on the apical surface of the polar cells ( M ). Inhibition of Rac1 ( N ), S1P receptor ( P ), and S1P biosynthesis ( Q ) cause a significant reduction in Rac1-GTP staining. In contrast, inhibition of mTOR ( O ), lack of glucose during culture ( R ), or inhibition of specific arms of glucose metabolism (PPP and HBP) ( S-T ) has no effect on Rac1-GTP levels or localization, suggesting a S1P dependent and glucose independent requirement for apical Rac1-GTP localization and mTOR activation.

Article Snippet: Various chemical drugs were purchased as indicated: Lectin from Triticum vulgaris (Sigma #L0636, 20µg/ml), YZ9 (Cayman Chemical #15352, 1µM), ST045849 (TimTec, 0.8µM), Tunicamycin (Sigma #T7765, 5µM), 6-Aminonicotinamide (6-AN) (Sigma #A68203, 2µM), N-Acetylcysteine (NAC) (Sigma #A7250, 1mM), Hypotaurine (Sigma #H1384, 1mM), Shikonin (Cayman Chemical #14751, 1µM), Azaserine (Sigma #A4142, 1µM), DON (Sigma #D2141, 20µM), Aphidicolin (Cayman Chemical #14007, 5µM), α-Amanitin (Cayman Chemical #17898, 100µg/ml), Cycloheximide (Cayman Chemical #14126, 10µg/ml), Torin2 (Cayman Chemical #14185, 10nM), INK128 (Cayman Chemical #11811, 100nM), JTE-013 (TOCRIS #2392, 1µM), SKI-II (Echelon #B-0024, 0.8µM), NSC26722 (TOCRIS #2161, 100µM), BYL719 (Cayman Chemical #16986, 1µM), BKM120 (Cayman Chemical #11587, 0.5µM), GDC0941 (Cayman Chemical #11600, 0.5µM), TGX221 (Cayman Chemical #10007349, 0.5µM), BX912 (Cayman Chemical #14708, 100nM), GSK2334470 (Cayman Chemical #18095, 50nM), MK2206 (Cayman Chemical #11593, 200nM).

Techniques: Expressing, Quantitation Assay, Blocking Assay, Western Blot, Activation Assay, Staining, Inhibition

Journal: bioRxiv

Article Title: Glucose metabolism distinguishes TE from ICM fate during mammalian embryogenesis

doi: 10.1101/826875

Figure Lengend Snippet: Embryos are isolated at 18h and cultured in either +G or –G media until 78h, and inhibitors were added to the medium at 50h. Cell membranes are marked by phalloidin-FITC staining (green). ( A-D ) mTOR target expression requires glucose. In control embryos high levels of pS757-ULK1 ( A ) or pS236-RPS6 ( C ) are seen at 78h. Embryos cultured in –G medium show a dramatic decrease in the levels of pS757-ULK1 ( B ) and pS236-RPS6 ( D ). ( E-H ) CDX2 expression is lost upon mTOR inhibition with Torin2 ( F ) and INK128 ( G ) and inhibition of S1P signaling by JTE-013 ( H ). ( I-P ) Inhibition of mTORC1 and S1P signaling does not perturb OCT4 and YAP1 expression. mTOR inhibition with Torin2 ( J, N ) and INK128 ( K, O ) and S1P signaling by JTE-013 ( L, P ) does not affect OCT4 ( I-L ) and YAP1 ( M-P ). ( Q ) Quantitation of phosphorylation of 4E-BP1 in embryos in which the mTOR and S1P pathways are inhibited (the control data are shared with ).

Article Snippet: Various chemical drugs were purchased as indicated: Lectin from Triticum vulgaris (Sigma #L0636, 20µg/ml), YZ9 (Cayman Chemical #15352, 1µM), ST045849 (TimTec, 0.8µM), Tunicamycin (Sigma #T7765, 5µM), 6-Aminonicotinamide (6-AN) (Sigma #A68203, 2µM), N-Acetylcysteine (NAC) (Sigma #A7250, 1mM), Hypotaurine (Sigma #H1384, 1mM), Shikonin (Cayman Chemical #14751, 1µM), Azaserine (Sigma #A4142, 1µM), DON (Sigma #D2141, 20µM), Aphidicolin (Cayman Chemical #14007, 5µM), α-Amanitin (Cayman Chemical #17898, 100µg/ml), Cycloheximide (Cayman Chemical #14126, 10µg/ml), Torin2 (Cayman Chemical #14185, 10nM), INK128 (Cayman Chemical #11811, 100nM), JTE-013 (TOCRIS #2392, 1µM), SKI-II (Echelon #B-0024, 0.8µM), NSC26722 (TOCRIS #2161, 100µM), BYL719 (Cayman Chemical #16986, 1µM), BKM120 (Cayman Chemical #11587, 0.5µM), GDC0941 (Cayman Chemical #11600, 0.5µM), TGX221 (Cayman Chemical #10007349, 0.5µM), BX912 (Cayman Chemical #14708, 100nM), GSK2334470 (Cayman Chemical #18095, 50nM), MK2206 (Cayman Chemical #11593, 200nM).

Techniques: Isolation, Cell Culture, Staining, Expressing, Inhibition, Quantitation Assay

Journal: iScience

Article Title: OsFKBP12 transduces the sucrose signal from OsNIN8 to the OsTOR pathway in a loosely binding manner for cell division

doi: 10.1016/j.isci.2024.111555

Figure Lengend Snippet:

Article Snippet: Torin2 , Immunoway , Cat# MC0120.

Techniques: Binding Assay, Virus, Recombinant, Magnetic Beads, SYBR Green Assay, Luciferase, Lysis, Mutagenesis, CRISPR, Knock-Out, Over Expression, Knockdown, Plasmid Preparation, Software, Real-time Polymerase Chain Reaction

Journal: iScience

Article Title: OsFKBP12 transduces the sucrose signal from OsNIN8 to the OsTOR pathway in a loosely binding manner for cell division

doi: 10.1016/j.isci.2024.111555

Figure Lengend Snippet:

Article Snippet: Four inhibitors, AZD8055 (Macklin, A837230), Torin 2 (Immunoway, MC0120), Rapamycin (Biorbyt, orb61092) and FK506 (abcam, ab120223), all were hydrophobic and required to dissolve in dimethyl sulfoxide (DMSO), but concentration of DMSO in the medium greater than 10 μL/mL would inhibit growth of sprout during germination in rice ( ).

Techniques: Binding Assay, Virus, Recombinant, Magnetic Beads, SYBR Green Assay, Luciferase, Lysis, Mutagenesis, CRISPR, Knock-Out, Over Expression, Knockdown, Plasmid Preparation, Software, Real-time Polymerase Chain Reaction