Journal: Molecular Oncology

Article Title: Sphingosine‐1‐phosphate‐lyase deficiency affects glucose metabolism in a way that abets oncogenesis

doi: 10.1002/1878-0261.13300

Figure Lengend Snippet: Remodeled glucose metabolism in SGPL1‐deficient MEFs. (A) Colorimetric determination of G6P shows increased levels in KO (red bars) relative to WT (white bars) MEFs. (B–D) Protein quantification of GLUT1, PFK, GAPDH, LDH, PDH, and IDH and HIF‐1 in WT controls (white bars) and SGPL1‐deficent (KO) MEFs (red bars). (E) Rates of proliferation of KO (red bars) are increased relative to WT (white bars) MEFs. (F) Protein quantification of GLUT1, PFK, and GAPDH in WT MEFs cultured in the absence or presence of 10 n m FTY720 (FTY) for 24 h. Shown is one representative western blot for each protein. β‐Actin was used as loading control. Bars represent means ± SEM ( n ≥ 3, **** P < 0.00005, *** P < 0.0005, ** P < 0.005, * P < 0.05; unpaired student t ‐test). a.u., arbitrary units; HIF‐1, hypoxia‐inducible factor 1.

Article Snippet: Glucose‐6‐phosphate was determined by colorimetric detection at 450 nm using the G6P Assay kit (Merck; MAK014).

Techniques: Cell Culture, Western Blot

Journal: bioRxiv

Article Title: A novel non-catalytic scaffolding activity of Hexokinase 2 contributes to EMT and metastasis

doi: 10.1101/2021.04.08.439049

Figure Lengend Snippet: a. Nickel-coated 96-well plates were incubated overnight at 4°C with His-PRKAR1a (50 nM) and then incubated with Myc-HK2 (0.25– 64 nM) in blocking buffer or with blocking buffer without HK2. HK2 binding was detected with anti-Myc-HRP conjugated antibody, and an HRP-catalyzed reaction with a chromogenic substrate solution. Right panel: Nickel-coated plates were incubated with His- PRKAR1a (50 nM) overnight and then with Myc-HK2 (32 nM) for 2h. Thirty minutes before the end of the later incubation, increasing concentrations of G6P (0 – 100uM) were added to the wells and detection was carried out as above. b. Glutathione-coated 96-well plates were incubated overnight at 4°C with GST-GSK3b (50 nM) and then incubated with Myc-HK2 (0.25– 64 nM) in blocking buffer or with blocking buffer without HK2. HK2 binding was detected as described above. Right panel: Glutathione -coated plates were incubated with GST-GSK3b (50 nM) overnight and then with Myc-HK2 (32 nM) for 2h. Thirty minutes before the end of the later incubation, increasing concentrations of G6P (0 – 100uM) were added to the wells and detection was carried out as above. c. Nickel-coated 96-well plates were incubated overnight at 4°C with His-PRKAR1a (50 nM) or with blocking buffer in absence of PRKAR1a, and then incubated with Myc-HK2 (32 nM) in blocking buffer or with blocking buffer without HK2 for 2h. GST-GSK3b or blocking buffer without GSK3b was then added for 2h. GSK3b binding was detected with anti-GST-HRP conjugated antibody, and an HRP-catalyzed reaction with a chromogenic substrate solution. Right panel: Nickel -coated plates were successively incubated with His-PRKAR1a (50 nM) overnight, HK2-Myc (32nM) for 2h and then GST-GSK3b (32 nM) for 2h. Thirty minutes before the end of the later incubation, increasing concentrations of G6P (0 – 1000uM) were added to the wells and detection was carried out as above. Results are the mean ± SEM of 3 independent experiments. e. Nickel-coated plates were incubated overnight at 4°C with His-HK2 (50 nM) and then successively incubated with Myc-PRKAR1α (32nM), PKAc (20nM) and GST-GSK3β (32nM). For the negative control experiments, His-HK2 or Myc-PRKAR1α were omitted. Once all proteins are bound, antibodies against HK2, PRKAR1α, PKAc or GSK3β are added for overnight incubation and detection is carried out after incubation with HRP-conjugated anti-rabbit or mouse IgG as described in Methods. f. Nickel-coated plates were incubated overnight at 4°C with His-HK2 (50 nM) and then successively incubated with Myc-PRKAR1α (32nM), PKAc (20nM) and GST-GSK3β (32nM). Kinase buffer in presence or absence of cAMP is added to appropriate wells and P-GSK3β antibody is added overnight, and then detected by incubation with HRP-conjugated anti-rabbit IgG. Bound total GSK3β is detected with anti-GST-HRP conjugated antibody. Both detections were revealed by HRP-catalyzed reaction with a chromogenic substrate solution. For G6P inhibition, G6P was added 30 minutes before the end of the incubation with GST-GSK3β.

Article Snippet: Importantly, as we had previously suggested the feedback inhibition of HK2 by its own catalytic product G6P could be used as a strategy to target HK2 for cancer therapy by developing mimetics of G6P or 2DG6P to inhibit its activity .

Techniques: Incubation, Blocking Assay, Binding Assay, Negative Control, Inhibition

Journal: bioRxiv

Article Title: A novel non-catalytic scaffolding activity of Hexokinase 2 contributes to EMT and metastasis

doi: 10.1101/2021.04.08.439049

Figure Lengend Snippet: a. Schematic showing the effect of DHEA, 6-AN, and the knockdown of 6PGDH on the PPP and glycolysis. b. Hela cells were treated with either DMSO, 6-AN or DHEA. At the indicated time points, cells were harvested for immunoblotting using anti-p-GSK3β and anti-GSK3 α/β . c. Intracellular levels of G6P in control (DMSO treated), 200μM 6-AN treated, and 200μM DHEA treated cells at different time points after treatment. Results are the mean ± SEM of 3 independent experiments in triplicate. *p<0.05 d. Upper panel: Simplified schematic of steps in glycolysis and the pentose phosphate pathway (PPP), showing 13 C labelling patterns resulting from [1,2- 13 C 2 ]-glucose substrate and the conversion to M+1 and M+2 lactate. Red filled circles indicate 13 C atoms. Abbreviations: G6P, glucose-6-phosphate; 6PGL, 6-phosphogluconolactone; 6PG, 6-phosphogluconate; Ru5P, ribulose-5-phosphate; F6P, fructose-6-phosphate; G3P, glyceraldehyde-3-phosphate; PEP, phosphoenolpyruvate; PYR, pyruvate. Bottom panels: [1, 2- 13 C 2 ]-glucose metabolic labelling in A549 cells showing the effect of 6-AN or the DOX-inducible knockdown of 6PDGH on the PPP and glycolysis (cells were treated with either 6-AN for 16h or with DOX for 4 days). Results show relative abundance of intracellular 1x 13 C lactate (M+1) and 2x 13 C lactate (M+2) after 25 mM [1, 2- 13 C 2 ]-glucose labeling for 4 h. Results are the mean ± SEM of 3 independent experiments. *p<0.05, ***p<0.01. e. A549 cells stably expressing each shRNA in the tet-on system were treated with doxycycline (DOX, 0.2ug/ml) for six days, cells were harvested and analyzed by immunoblotting. A549 cells expressing inducible 6PGD shRNA (Tet-6PGDsh) were also treated with 6-AN for 16h in the absence of DOX. f. Cells were treated and isotopically labelled as in d, and 6PG level was measured after GPI and 6PGD knockdown or after treatment with 6-AN. Relative abundance of intracellular 2x 13 C G6P (M+2) and 2x 13 C 6PG (M+2) after 25 mM [1, 2- 13 C 2 ]-glucose labeling for 4 h is shown. Results are the mean ± SEM of 3 independent experiments g. Cells were treated and isotopically labelled as in d, and G6P level was measured after GPI and 6PGD knockdown or after treatment with 6-AN. h. A549 cells expressing inducible 6PGD sh RNA (Tet-6PGDsh) were exposed to DOX and pGSK3 and NRF2 levels were followed for 4 days after DOX addition (Numbers indicate level relative to tubulin).

Article Snippet: Importantly, as we had previously suggested the feedback inhibition of HK2 by its own catalytic product G6P could be used as a strategy to target HK2 for cancer therapy by developing mimetics of G6P or 2DG6P to inhibit its activity .

Techniques: Knockdown, Western Blot, Control, Metabolic Labelling, Labeling, Stable Transfection, Expressing, shRNA

Journal: bioRxiv

Article Title: A novel non-catalytic scaffolding activity of Hexokinase 2 contributes to EMT and metastasis

doi: 10.1101/2021.04.08.439049

Figure Lengend Snippet: When cells have high glucose flux, HK2 brings PKA and GSK3 into close proximity. In the presence of cAMP, PKA is released from PRKAR1a to phosphorylate GSK3. When glucose flux is attenuated and G6P accumulates, an allosteric change is conferred to HK2 releasing GSK3 and PRKAR1a and increasing the availability of phosphorylated GSK3 to PP2A. It is possible that the amino-terminus half and the carboxy- terminus half of HK2 each binds GSK3 and PRKAR1a.

Article Snippet: Importantly, as we had previously suggested the feedback inhibition of HK2 by its own catalytic product G6P could be used as a strategy to target HK2 for cancer therapy by developing mimetics of G6P or 2DG6P to inhibit its activity .

Techniques:

Journal: Infection and Drug Resistance

Article Title: Effect of Colistin, Fosfomycin and Meropenem/Vaborbactam on Carbapenem-Resistant Enterobacterales in Egypt: A Cross-Sectional Study

doi: 10.2147/IDR.S385411

Figure Lengend Snippet: Antimicrobial Susceptibility Pattern of the Uropathogenic CRE Strains

Article Snippet: Fosfomycin susceptibility was tested using the disc diffusion method with fosfomycin 200 μg disc containing glucose-6-phosphate (G6P) (Liofilchem, Roseto Degli Abruzzi, Italy) on Mueller Hinton agar and interpreted using CLSI guidelines, where ≥16 mm was sensitive, 13–15 mm intermediate and ≤12 mm resistant.

Techniques:

Journal: Infection and Drug Resistance

Article Title: Effect of Colistin, Fosfomycin and Meropenem/Vaborbactam on Carbapenem-Resistant Enterobacterales in Egypt: A Cross-Sectional Study

doi: 10.2147/IDR.S385411

Figure Lengend Snippet: Meropenem/vaborbactam and fosfomycin susceptibility testing using the MIC Test Strip according to CLSI guidelines. ( A ) Meropenem/vaborbactam susceptible strain (MIC = 0.032 µg/mL), ( B ) meropenem/vaborbactam resistant strain (MIC > 256 µg/mL), ( C ) fosfomycin susceptible strain (MIC = 0.25 µg/mL) and ( D ) fosfomycin resistant strain (MIC > 256 µg/mL).

Article Snippet: Fosfomycin susceptibility was tested using the disc diffusion method with fosfomycin 200 μg disc containing glucose-6-phosphate (G6P) (Liofilchem, Roseto Degli Abruzzi, Italy) on Mueller Hinton agar and interpreted using CLSI guidelines, where ≥16 mm was sensitive, 13–15 mm intermediate and ≤12 mm resistant.

Techniques: Stripping Membranes

Journal: Infection and Drug Resistance

Article Title: Effect of Colistin, Fosfomycin and Meropenem/Vaborbactam on Carbapenem-Resistant Enterobacterales in Egypt: A Cross-Sectional Study

doi: 10.2147/IDR.S385411

Figure Lengend Snippet: Antibiotic Resistance Pattern and the Distribution of Integron and fosA Genes Among Carbapenemase Producing Enterobacterales (CPE) and Non-CPE Isolates

Article Snippet: Fosfomycin susceptibility was tested using the disc diffusion method with fosfomycin 200 μg disc containing glucose-6-phosphate (G6P) (Liofilchem, Roseto Degli Abruzzi, Italy) on Mueller Hinton agar and interpreted using CLSI guidelines, where ≥16 mm was sensitive, 13–15 mm intermediate and ≤12 mm resistant.

Techniques: