Journal: Frontiers in Molecular Biosciences

Article Title: Butyrate Suppresses Glucose Metabolism of Colorectal Cancer Cells via GPR109a-AKT Signaling Pathway and Enhances Chemotherapy

doi: 10.3389/fmolb.2021.634874

Figure Lengend Snippet: Butyrate suppressed G6PD abundance and DNA synthesis of colon cancer cells. (A) The concentration of intracellular metabolites associated with glucose metabolism in HCT116 cells being incubated with 2 mM butyrate (labeled as “NaB”) or PBS vehicle (marked as “Con”) for 24 h were measured by liquid chromatograph-mass spectrometer/mass spectrometer (LC-MS/MS). (B) The variation of central metabolites in HCT116 cells being incubated with 2 mM butyrate or PBS vehicle for 24 h was annotated in the schematic diagram of glycolysis pathway. (C) After HCT116 cells were incubated with 2 mM butyrate for 24 h, the expression levels of genes that regulate glucose metabolism were analyzed by qPCR. ( D) The protein levels of G6PD in HCT116 and LoVo cells after being treated with 2 mM butyrate for 24 h were tested by western blotting. (E) After incubating HCT116 and LoVo cells with 2 mM butyrate or PBS vehicle for 24 h, DNA synthesis efficiency in both cell lines was measured by BrdU incorporation assay. The error bars showed standard deviations from independent triplicates. * p < 0.05, ** p < 0.01, *** p < 0.001. G6P, D-Glucose 6-phosphate; F6P, Beta-D-Fructose 6-phosphate; F1,6BP, D-Fructose 1,6-bisphosphate; PEP, Phosphoenolpyruvate; PYR, Pyruvate; LAC, Lactate; R5P, Ribose-5-phosphate.

Article Snippet: Antibodies against GLUT1 (1:1000) and G6PD (1:1000) were purchased from Cell Signaling Technology (United States).

Techniques: DNA Synthesis, Concentration Assay, Incubation, Labeling, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Expressing, Western Blot, BrdU Incorporation Assay

Journal: Frontiers in Molecular Biosciences

Article Title: Butyrate Suppresses Glucose Metabolism of Colorectal Cancer Cells via GPR109a-AKT Signaling Pathway and Enhances Chemotherapy

doi: 10.3389/fmolb.2021.634874

Figure Lengend Snippet: Butyrate regulated G6PD abundance and DNA synthesis of colon cancer cells through the AKT pathway. (A ) The protein levels of G6PD were measured by western blotting in HCT116 and LoVo cells being treated with 2 mM butyrate alone or co-incubated with 2 mM butyrate and 10.96 μM SC79 for 24 h. (B) The effects of 10.96 μM SC79 (marked as “NaB + SC79”) or PBS vehicle (labeled as “NaB + Con”) on cell proliferation were tested by CCK-8 assay in HCT116 and LoVo cells which were incubated with 2 mM butyrate for 24 h. (C) After being treated with a combination of 2 mM butyrate and PBS vehicle (labeled as “NaB + Con”) or 2 mM butyrate and 10.96 μM SC79 (marked as “NaB + SC79”) for 24 h, the DNA synthesis efficiency in HCT116 and LoVo cells was assayed by BrdU incorporation experiment. The error bars showed standard deviations from independent triplicates. * p < 0.05, *** p < 0.001.

Article Snippet: Antibodies against GLUT1 (1:1000) and G6PD (1:1000) were purchased from Cell Signaling Technology (United States).

Techniques: DNA Synthesis, Western Blot, Incubation, Labeling, CCK-8 Assay, BrdU Incorporation Assay

Journal: Molecular Medicine Reports

Article Title: Aspirin inhibits glucose-6-phosphate dehydrogenase activity in HCT 116 cells through acetylation: Identification of aspirin-acetylated sites

doi: 10.3892/mmr.2016.5449

Figure Lengend Snippet: Aspirin-mediated acetylation of glucose-6-phosphate dehydrogenase (G6PD) is greater in (A) HCT 116 cells compared with in (B) HT-29 cells. Subconfluent cells were left untreated or were treated with aspirin for 24 h, lysates were prepared, and equal amounts of protein were immunoprecipitated with rabbit agarose-conjugated anti-acetyl lysine antibody. Agarose-bound proteins were eluted and immunoblotted with anti-G6PD antibody. (C) HCT-116 and (D) HT-29 samples were immunoblotted with anti-G6PD antibody. The experiments were repeated three times.

Article Snippet: The recombinant G6PD protein (isoform a ) was obtained from Origene Technologies, Inc. (Rockville, MD, USA).

Techniques: Immunoprecipitation

Journal: Molecular Medicine Reports

Article Title: Aspirin inhibits glucose-6-phosphate dehydrogenase activity in HCT 116 cells through acetylation: Identification of aspirin-acetylated sites

doi: 10.3892/mmr.2016.5449

Figure Lengend Snippet: Effects of aspirin on glucose-6-phosphate dehydrogenase (G6PD) activity in HCT 116 and HT-29 cells. Cells were cultured and left untreated or were treated with aspirin for 24 h. Protein (100 µ g) was used to conduct a G6PD assay. The reaction mixture was incubated at 37°C for 30 min, and absorbance was measured at 450 nm. G6PD activity was expressed as a percentage of control. The experiments were repeated three times. Data are represented as mean ± standard deviation. * P<0.05, ** P<0.01, *** P<0.001 vs. the control.

Article Snippet: The recombinant G6PD protein (isoform a ) was obtained from Origene Technologies, Inc. (Rockville, MD, USA).

Techniques: Activity Assay, Cell Culture, G6PD Assay, Incubation, Standard Deviation

Journal: Molecular Medicine Reports

Article Title: Aspirin inhibits glucose-6-phosphate dehydrogenase activity in HCT 116 cells through acetylation: Identification of aspirin-acetylated sites

doi: 10.3892/mmr.2016.5449

Figure Lengend Snippet: Mass spectrometry (MS) analysis of recombinant glucose-6-phosphate dehydrogenase (G6PD) isoform a . In vitro acetylation of recombinant G6PD by aspirin. (A) A total of 5 ng in vitro acetylated recombinant G6PD was immunoblotted with anti-acetyl lysine antibody and the protein band was detected by enhanced chemiluminescence. (B-D) MS/MS fragmentation spectra showing acetyl modification of (B) K77, (C) K201 and (D) K235.

Article Snippet: The recombinant G6PD protein (isoform a ) was obtained from Origene Technologies, Inc. (Rockville, MD, USA).

Techniques: Mass Spectrometry, Recombinant, In Vitro, Tandem Mass Spectroscopy, Modification

Journal: Molecular Medicine Reports

Article Title: Aspirin inhibits glucose-6-phosphate dehydrogenase activity in HCT 116 cells through acetylation: Identification of aspirin-acetylated sites

doi: 10.3892/mmr.2016.5449

Figure Lengend Snippet: Location of aspirin-acetylated lysine residues identified in the present study and the naturally acetylated lysines on G6PD ( 25 ).

Article Snippet: The recombinant G6PD protein (isoform a ) was obtained from Origene Technologies, Inc. (Rockville, MD, USA).

Techniques:

Journal: Molecular Medicine Reports

Article Title: Aspirin inhibits glucose-6-phosphate dehydrogenase activity in HCT 116 cells through acetylation: Identification of aspirin-acetylated sites

doi: 10.3892/mmr.2016.5449

Figure Lengend Snippet: 3-Dimension space-filling model of recombinant glucose-6-phosphate dehydrogenase (G6PD; NP_000393), is shown. The location of aspirin-acetylated lysine residues are highlighted in blue (K77, K112, K119, K201, K235, K390, K396, K416, K438, K459, K462, K527, K538, K544).

Article Snippet: The recombinant G6PD protein (isoform a ) was obtained from Origene Technologies, Inc. (Rockville, MD, USA).

Techniques: Recombinant