Journal: Cell Death & Disease

Article Title: Tumor-suppressive function of EZH2 is through inhibiting glutaminase

doi: 10.1038/s41419-021-04212-7

Figure Lengend Snippet: A, B Immunoblotting for cleaved-PARP and GLS expression in HCT116 ( A ) or SW480 ( B ) cells pretreated with or without GSK126 (4 μM, 48 h) or/and BPTES (10 μM, 48 h), followed by glucose deprivation treatment (0 mM, 16 h). Actin is used as a loading control. C, D Immunoblotting for cleaved-PARP and GLS expression in HCT116 NC and shEZH2 cells ( C ) or SW480 NC and shEZH2 cells ( D ) cells pretreated with DMSO or BPTES (10 μM, 48 h), followed by glucose deprivation treatment (0 mM, 16 h). E Three-day growth of HCT116 NC and shEZH2 cells or SW480 NC and shEZH2 cells in presence or not of 5 μM BPTES. F Growth curves of HCT116 or SW480 cells in presence or not of 2 μM GSK126 or/and 5 μM BPTES. G, H Colony formation of HCT116 ( G ) or SW480 ( H ) cells in presence or not of 2 μM GSK126 or/and 5 μM BPTES. The experiments in A–D and G , H were repeated twice independently with similar results. In E , F , data are mean ± s.d., n = 3 independent experiments; P -values were calculated using two-tailed unpaired Student’s t -test or two-way ANOVA test. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The primary antibodies and concentrations used for Western blotting were following: EZH2 (1:1000, 5246 S, Cell Signaling Technology, Danvers, MA, USA), H3K27me3 (1:1,000, 9733 S, Cell Signaling Technology), C-PARP (1:2,000, 5625 S, Cell Signaling Technology), GLS (1:1,000, 56750 T, Cell Signaling Technology), Actin (1:10,000, AP0060, Bio-world, Nanjing, China).

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

Journal: Molecules (Basel, Switzerland)

Article Title: Atractylenolide III Ameliorates Bile Duct Ligation-Induced Liver Fibrosis by Inhibiting the PI3K/AKT Pathway and Regulating Glutamine Metabolism.

doi: 10.3390/molecules28145504

Figure Lengend Snippet: Figure 8. Inhibition of glutaminase by ATL III in BDL-induced liver fibrosis. (A) Molecular docking of ATL III with the protein GLS1. (B) ATL III binding to the GLS1. (C) Glutamate content in the liver. (D) Representative GLS1 and GLS2 immunohistochemical images of liver tissue sections. (E) Protein expression of GLS1 and GLS2 in the liver. Protein expression statistics of (F) GLS1 and (G) GLS2. Results are expressed as mean ± SD, n = 3. ## p < 0.01 compared with the sham group. * p < 0.05, ** p < 0.01 compared with the model group.

Article Snippet: Antibodies against GLS1 (A3885) and GLS2 (A16029) were purchased from ABclonal Technology (Wuhan, China).

Techniques: Inhibition, Binding Assay, Immunohistochemical staining, Expressing

Journal: Molecules (Basel, Switzerland)

Article Title: Atractylenolide III Ameliorates Bile Duct Ligation-Induced Liver Fibrosis by Inhibiting the PI3K/AKT Pathway and Regulating Glutamine Metabolism.

doi: 10.3390/molecules28145504

Figure Lengend Snippet: Figure 9. ATL III affects ECM synthesis through the PI3K/AKT signaling pathway, regulates glutamine metabolism to inhibit GLS1, and improves metabolic disorders, thereby ameliorating liver fibrosis.

Article Snippet: Antibodies against GLS1 (A3885) and GLS2 (A16029) were purchased from ABclonal Technology (Wuhan, China).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Glutamine metabolism is essential for coronavirus replication in host cells and in mice

doi: 10.1016/j.jbc.2024.108063

Figure Lengend Snippet: GLS expression is increased during coronavirus infection. A , Whole-cell lysates of uninfected HBECs or HBECs infected with HCoV-OC43 (MOI 0.01, 24 h) were analyzed for viral N-protein OC43, GLS, and GLS2 expression levels by Western blot. B , similar experiment as ( A ) in HCT8 cells. C , total RNA was isolated from uninfected HBECs or HBECs infected with HCoV-OC43 (MOI 0.01, 24 h). qPCR assays showed GLS and GLS2 mRNA levels before and after HCoV-OC43 infections (n = 3). D , qPCR assays performed in HCT8 cells as in ( C ) (n = 4). E , co-immunofluorescent staining with HCoV-OC43 and GLS antibodies in HBECs. Cells were grown in 4-well slide chambers for 24 h and infected with the HCoV-OC43 virus for 24 h. One drop per well of NucBlue Live Cell stain solution was added to each well before the cells were fixed with formaldehyde (3.7%) (a) nuclear staining with DAPI ( blue ), (b) staining with GLS rabbit polyclonal antibody and goat anti-rabbit Alexa Fluor 568 secondary antibody ( red ), (c) staining with HCoV-OC43 mouse monoclonal antibody and goat anti-mouse Alexa Fluor 488 secondary antibody ( green ). (d) Merging of the images described in a,b,c. F , Western blot analysis shows the time course of GLS and HCoV-OC43 expression levels during virus infection in HBECs. G , qPCR assays for GLS levels were performed on total RNA samples isolated at different time points of virus-infected HBECs (n = 3). H , qPCR assays for HCoV-OC43 levels were performed on the total RNA samples isolated at different time points of virus-infected media in HBECs (n = 3). I , whole cell lysates of MRC5 cells uninfected or infected with HCoV-229E (MOI 0.01, 24h) were analyzed by Western blot for GLS and GLS2 expression levels.

Article Snippet: The GLS antibody was raised against the sequence-KLDPRREGGDQRHS and GLS2 antibody was obtained from ProSci (Cat. 6217).

Techniques: Expressing, Infection, Western Blot, Isolation, Staining, Virus

Journal: The Journal of Biological Chemistry

Article Title: Glutamine metabolism is essential for coronavirus replication in host cells and in mice

doi: 10.1016/j.jbc.2024.108063

Figure Lengend Snippet: Schematic of GLS and GLS2 mediated coronavirus replication. Coronaviruses hijack the metabolic program of host cells by increasing the expression of glutaminase enzymes. Two possible mechanisms for upregulating GLS upon beta-coronavirus infection involve signaling through c-Myc and c-Jun. Alpha-coronavirus replication occurs by upregulating GLS2 expression (HCoV-229E in MRC5 cells).

Article Snippet: The GLS antibody was raised against the sequence-KLDPRREGGDQRHS and GLS2 antibody was obtained from ProSci (Cat. 6217).

Techniques: Expressing, Infection

Journal: Advanced Science

Article Title: Overcoming Cancer Persister Cells by Stabilizing the ATF4 Promoter G‐quadruplex

doi: 10.1002/advs.202401748

Figure Lengend Snippet: Glutamine‐restrictive therapy benefits from the low levels of ATF4. A) Tissue microarray of NSCLC patients with tissue immunofluorescence assay. Representative tissue immunofluorescence images with ATF4 (Red), GS (Green), and GLS (Pink). White arrows indicate GLS filaments. Scale bar: 10 µm. B) Correlation analysis between the number of relative GLS filament structures and positive cell density of ATF4 in the tissue microarray. C) Correlation analysis between positive cell density of GS and ATF4 in the tissue microarray. D) Schematic of tumors after intratumoral injection of shATF4 lentivirus with glutamine‐deficient diet and 2% glutamine diet in the established xenograft model ( n = 6 per group). E) Tumor images ( n = 6 per group). F) Tumor volume, body weight, and tumor weight of nude mice with intratumoral injection of shATF4 lentivirus and different glutamine diets ( n = 6 per group). G) Protein expression levels in xenograft tumors after intratumoral injection of shATF4 lentivirus and different glutamine diets ( n = 3 independent experiments). H) Relative glutamine content in different glutamine diet groups ( n = 6 independent experiments). Glutamine concentrations of 2% and 0% glutamine diet are represented by 2%Q and ‐Q, respectively. Data are shown as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Data were analyzed using two‐tailed Student's t‐tests (F, G, H) in GraphPad Prism 9.5.0. Source unprocessed blots are provided.

Article Snippet: Subsequently, the tissue microarray was incubated with anti‐ATF4 (Abcam, ab184909, 1:200), GS (Affinity, DF7341, 1:200), and GLS (Proteintech, 23549‐1‐AP, 1:200) antibodies overnight at 4 °C.

Techniques: Microarray, Immunofluorescence, Injection, Expressing, Two Tailed Test