Fructose 1 6 bisphosphate

Hexokinase, glucose-6-phosphate dehydrogenase, glucose-6-phosphatase, fructose-1-6-bisphosphate were purchased from Sigma Aldrich (India). Blood glucose, lipid profile, and antioxidant enzymes kits were purchased from Span Diagnostics, Surat (India). Streptozotocin was purchased from Sigma Aldrich (India). Other needed chemicals used in the study were procured either from Sigma Aldrich (India) or CDH Mumbai (India).

Pyruvate kinase activity assay was previously described [30 (link),31 (link)]. The tissues were lysed in RIPA buffer (150 mM NaCl; 10 mM Tris, pH 7.2; 0.1% SDS; 1% Triton X-100; 1% sodium deoxycholate; 5 mM EDTA). The lysates were centrifuged at 15,920× g at 4°C for 30 min. Equal amounts of protein (1 μg) were incubated with 100 mM KCl (Duksan Pure Chemicals Co.), 50 mM Tris (Amresco, Cleveland, OH) pH 7.5, 5 mM, 0.6 mM ADP, 0.5 mM phosphoenolpyruvate (Sigma-Aldrich), 10 μM fructose-1,6-bisphosphate (Sigma-Aldrich), 240 μM NADH (Sigma-Aldrich), and 8 units of lactate dehydrogenase (Sigma-Aldrich). The absorbance change of NADH’s oxidation was measured at 320 nm using a FLUOstar OPTIMA luminometer (BMG Labtech, Offenburg, Germany).

Etoposide (VP16) was purchased from Calbiochem. Fructose 1, 6-bisphosphate (FBP), phosphoenolpyruvic acid (PEP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), hygromycin B were purchased from Sigma. Rabbit polyclonal antibodies against PKM2, H2AX, γ-H2AX, ATM, Phospho-ATM (Ser1981), ATR, Phospho-ATR (Ser428), DNAPK and phospho-DNAPK (Ser 2056), and β-actin were obtained from Cell Signaling Technology. Phospho-DNAPK (Ser 2609) antibody was purchased from Signal way Antibody (College Park). Goat antibody against LaminB was obtained from Santa Cruz Biotechnology (Santa Cruz). Mouse antibody for γ-H2AX were from Abcam. Monoclonal antibodies for His or GST were from Sigma.

2, 2-Diphenyl-1-pycrylhydrazyl, glucose 6-P dehydrogenase (EC 1.1.1.49), p-nitrophenyl-β-D-glucopyranoside (pNPG), CM-Chitin-RBV substrates, D-(+)-glucose, laminarin, 2-deoxy-Dglucose, fructose-1,6-bisphosphate, NADP, ATP, NADH, D-L-isocitrate, phenyl hydrazine-HCl, cystein-HCl, EDTA, cAMP Enzyme Immunoassay Kit (Cat no 100 CA201) were purchased from Sigma Chemical Company, USA. Other chemicals of analytical grade were purchased locally.

Cytosolic fructose-1,6-bisphosphatase activity was determined according to the method of Sharkey et al. (1991 (link)) with a slight modification. The basal portions of rice shoots harvested at the four-leaf stage were frozen at − 80 °C then milled with a mortar and pestle in 20 mM HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid)-NaOH (pH 7.5), 100 mM KCl, and 0.5 mM EDTA. Each extraction solution was centrifuged at 13,000×g and 4 °C for 10 min. The supernatant was added to a reaction mixture consisting of 100 mM HEPES-NaOH (pH 7.5), 100 mM KCl, 5 mM MgCl₂, 0.5 mM EDTA, 0.5 mM NADP, one unit of phosphoglucoisomerase (Sigma-Aldrich Corp., Tokyo, Japan), and two units of glucose-6-phosphate dehydrogenase. The reaction was initiated by adding 40 μM fructose-1,6-bisphosphate (Sigma-Aldrich Corp., Tokyo, Japan). It was then spectrophotometrically monitored at 340 nm for increases in NADPH concentration for 5 min after the start of the enzymatic reaction. The amount of product formed was calculated from the increase in absorbance using the NADPH extinction coefficient of 6220 L mol^− 1 cm^− 1.