Phospho-GCN2 (Thr899) Antibody - #AF8154

Figure 7 COP decreases glutamine deficiency-induced ATF4 levels and improves the efficacy of glutamine-restrictive therapy. A) Protocol of COP (100 mg·kg−1) administration in glutamine restriction therapy and establishment of H460 xenograft tumor model. B) Image of tumor removal after intraperitoneal injection of COP (100 mg·kg−1) (n = 6 per group). C) Growth curves of tumor volume (n = 6 per group). D) Tumor volume of nude mice with COP treatment and glutamine restriction diet (n = 6 per group). E) Immunoblotting of indicated proteins in xenograft tumors after COP and glutamine restriction diet therapy in vivo (n = 3 independent experiments). F) Determination of glutamine content in xenograft tumors with glutamine restriction (n = 6 independent experiments). G) Proposed model of COP targeting ATF4-G4 enhancing sensitivity to glutamine restriction therapy. All immunoblots are representative of three biological replicates that showed similar results. Glutamine concentrations of 2% and 0% glutamine diet are represented by 2%Q and -Q, respectively. COP: coptisine chloride. Data are shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data were analyzed by two-tailed Student's t-tests (D,F) and One-way ANOVA (E) in GraphPad Prism 9.5.0.

Figure 6. BCAA increase PPAR-α expression in a GCN2/ATF6 pathway-dependent manner. (A) Expression of p-GCN2, GCN2 and ATF6 in the presence of increasing concentrations of BCAA (0, 0.429 mM, 0.858 mM, 1.716 mM, 3.432 mM) by western blotting (n=6). (B) Expression of p-GCN2, GCN2 and ATF6 in the presence of increasing concentrations of BCKA (0, 0.429 mM, 0.858 mM, 1.716 mM, 3.432 mM) by western blotting (n=6). BCKA mixture is composed of αKIC, αKIV and αKMV (weight ratio, αKIC: αKIV: αKMV= 2:1:1). (C) NRVMs were treated with control siRNA and ATF6 siRNA. 48 h after transfection, expression of ATF6 was determined by western blotting (n=4). (D-E) ATF6 siRNA transferred NRVMs were treated with or without BCAA (3.432 mM) (n=6). (D) PPAR-α expression was determined by western blotting. (E) Expression of Acaa2, Acadm, Cd36 and Cpt1b by real-time PCR. (F-G) ATF6 siRNA transferred NRVMs were treated with or without BCKA (3.432 mM) (n=6). (F) PPAR-α expression was determined by western blotting. (G) Expression of Acaa2, Acadm, Cd36 and Cpt1b by real-time PCR. (C) Data were analyzed by Student’s t test. (A-B) and (D-G) Data were analyzed by one-way ANOVA, followed by a Bonferroni post-hoc test. * P<0.05, ** P<0.01. All values are presented as mean ± SEM.

Figure 4 Phosphorylation levels of p-GCN2 and p-SREBP1 in cell experiment detected using western blot A, C: western blot; western blots in A are control, 5%DGR, model, 10%DGR. in proper order and in C are control, model, 5%DGR, 10%DGR. B, D: quantitative comparison of western blot detection results. Control: conventional culture without intervention; Model: without drug intervention after modelling; 5%DGR: 5% medium-dose medicated serum + 10% blank serum after modelling; 10%DGR: 10% medium-dose medicated serum + 5 % blank serum. DGR: Dangua Fang; p-GCN2: phospho-general control non-derepressible 2 (n = 4); p-SREBP 1: phospho- sterol-regulatory element binding protein 1 (n = 6). Compared with control group: aP < 0.05; compared with model group: bP < 0.05, cP < 0.01.