Pyruvate assay kit

ATP production was detected using an ATP Bioluminescent Assay Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), according to the manufacturer’s instructions. Glucose consumption assay, lactic acid production, pyruvate, and lactate dehydrogenase (LDH) were performed with a Glucose Measurement Assay Kit (Rongsheng Biotech, Shanghai, China), Lactic Acid Assay Kit (Nanjing Jiancheng Bioengineering Institute), Pyruvate Assay Kit (Nanjing Jiancheng Bioengineering Institute), and LDH Assay Kit (Wanleibio), respectively, according to the manufacturer’s instructions.

Lactate, pyruvate, and acetyl-CoA levels were separately determined using a Lactate Assay Kit (Jiancheng Bioengineering Institute, Nanjing, China), Pyruvate Assay Kit (Jiancheng Bioengineering Institute), and an Acetyl-Coenzyme Assay Kit (Solarbio, Beijing, China) according to the manufacturer’s protocols. All experiments were performed at least three times.

L-serine deaminase activity was measured based on a method published by Wood et al. [31 (link)] with minor modifications. C. terniflora leaves were collected pre- and post-treatment with high level UV-B irradiation for 5 h with or without a subsequent 36 h incubation in the dark. The leaves (500 mg) were frozen and then homogenized on ice using a mortar and pestle in 5 mL of extraction buffer (0.1 M Tris-HCl (pH 7.4), 1 mM ethylenediaminetetraacetic acid disodium salt, and 0.1 mM 2- mercaptoethanol). The resulting homogenate was centrifuged at 13,000 × g for 30 min at 4 °C. Supernatants, 400 μL each, were collected, and mixed with 560 μL reaction buffer (20 mM Tris-HCl (pH 8.5), 20 μM pyridoxal phosphate, and 20 mM L-serine). Before adding the enzyme and substrate, the assay tubes were warmed to 37 °C. After adding the substrate, the reaction was allowed to proceed for 1 h and then was stopped using 0.2 mL of 100 % trichloroacetic acid. The pyruvate in the samples was quantified using a Pyruvate assay kit (Nanjing Jiancheng, Jiangsu, China). Protein concentrations were determined using the Bradford assay [26 (link)] with bovine serum albumin as the standard. A unit of L-SD activity was arbitrarily defined as the amount of enzyme necessary to form 1 μM pyruvate of 1 mg protein in 1 h under the above experimental conditions.

Cells were seeded in 10‐cm culture dishes 1 day before all measurements. Ten million cells were collected and then resuspended and homogenized in 1 mL PBS. The cell debris was eliminated by centrifugation. The supernatant was deproteinized with perchloric acid using Deproteinizing Sample Preparation Kit (K808‐200; BioVison, Milpitas, CA, USA) before measurements. Intracellular pyruvate, lactate, and triglycerol levels were measured using Pyruvate Assay Kit, Lactic Acid Assay Kit, and Triglycerol Assay Kit (A081, A019, and A110, respectively; Nanjing Jiancheng Bioengineering Institute, Nanjing, China) at 505, 530, and 510 nm, respectively. Acetyl‐CoA concentration was measured using an Acetyl‐Coenzyme A Assay Kit (MAK039; Sigma‐Aldrich, St. Louis, MO, USA) according to the manufacturer's instructions (λex = 535/λem = 587 nm), and data were normalized to assay buffer. Dual‐Wave Violet Spectrophotometer (LAMBDA Bio 20, Waltham, MA, USA) was applied to measure the absorbance and fluorescence. Experiments were performed according to the manufacturer's instructions using three independent biological repeats.