Trichloroacetic acid

Purified YenTc of Y. entomophaga WT and chi2-sfGFP were sent in solution to the FGCZ, which performed the mass spectrometry and the subsequent analysis of primary data. Proteomics samples were prepared by trichloroacetic acid precipitation, followed by trypsin digestion. For trichloroacetic acid precipitation, proteins were precipitated with trichloroacetic acid (Sigma-Aldrich) at a final concentration of 5% and washed with ice-cold acetone. Samples were then air dried and dissolved in 10 mM Tris and 2 mM CaCl₂, pH 8.2. Samples were then enzymatically digested using trypsin. These digested samples were dried and dissolved in 20 μl double distilled water with 0.1% formic acid. Samples were transferred to autosampler vials for liquid chromatography–tandem mass spectrometry analysis (LC–MS/MS). For each sample, 3 µl were injected on a nanoAcquity UPLC coupled to a Q-Exactive mass spectrometer (Thermo Fisher). The acquired MS data were converted to a Mascot Generic File format (.mgs files). Identification of proteins was performed using the Mascot search engine (Matrixscience). The spectra were searched against the Y. entomophaga protein database.
All protein identification results were visualized using the Proteome software ‘Scaffold’.

Lipid peroxidation of the algae was examined by determining the malondialdehyde (MDA) content [55 (link)]. Algal cells (25 mL) were collected by centrifugation (3000× g, 3 min) and stored at −80 °C before the analyses. The samples were homogenised in 5 mL of 10% trichloroacetic acid, and 1 mL of the homogenate was mixed with 2 mL of 0.5% thiobarbituric acid (Sigma-Aldrich) in 10% trichloroacetic acid (Sigma-Aldrich). The mixture was then incubated in a water bath at 95 °C for 40 min, cooled down to room temperature, and centrifuged at 4000× g for 15 min. The absorbance of the supernatant was recorded at 532 and 600 nm [56 (link)], and MDA content was calculated using an extinction coefficient of 155 mM⁻¹ [57 (link)]. The results were expressed as µmol MDA per unit of dry weight.
Dry weight was determined using cells collected on glass microfibre filters (0.7 μm, GVS, USA) that were dried in the dehydrator at 90 °C until constant weight.

Cells were seeded into a 96-well plate (Corning) at an optimal density to maintain final readouts in the linear range of the assay. After adhering overnight, the cells were treated zampanolide at the indicated concentrations in 0.5% DMSO (Fisher Scientific, Waltham, MA, USA) for 48 h. A separate time zero plate was fixed with 10% trichloroacetic acid (Sigma-Aldrich, St. Louis, MO, USA) to provide a readout of cellular density at the time of drug addition. After treatment, the cells were fixed with 10% trichloroacetic acid for a minimum of 1 h. The cells were then stained with sulforhodamine B dye (Sigma-Aldrich) for 30 min. Excess dye was washed off and the cellular-bound dye was solubilized in 200 µL of 10 mM Tris before reading the optical density at 560 nm on the Spectramax plate reader running SoftMax Pro 5.4 (Molecular Devices, San Jose, CA, USA). The percent growth or cytotoxicity of cells during the treatment period was determined as compared to the time zero plate (y = 0) and vehicle treated wells (y = 100) and replicates graphed with error bars representing SEM. Concentrations that caused a 50% inhibition of cell growth (GI₅₀) and total growth inhibition (TGI) were determined by non-linear regression analysis of the data using Prism (Graphpad, San Diego, CA, USA). Data are from 3 independent experiments each run in triplicate ± SEM.

The chemicals used in the present work were purchased from various companies. Sodium nitrite, potassium persulfate, dipotassium hydrogenphosphate, ferric chloride, monosodium dihydrogenphosphate, potassium ferricyanide, trichloroacetic acid, disodium hydrogenphosphate, Rutin, Mueller-Hinton agar (MHA), and Folin-Ciocalteu reagent were purchased from Merck (Darmstadt, Germany), gallic acid from Riedel-de-Haen (Seelze, Germany), ammonium molybdate, linoleic acid, ferrous chloride tetrahydrate and aluminum chloride from BDH Labs (Cambridge, England), DPPH (2,2-diphenyl-1-picrylhydrazyl) and ascorbic acid from MP Biomedicals (Illkirch, France), sodium acetate from Daejung (Siheung City, Korea), ammonium thiocyanate from Alfa-Aesar (Karlsruhe, Germany), amoxicillin from GlaxoSmithKline (Karachi, Pakistan) and trichloroacetic acid, TPTZ (2,4,6 tripyridyl-s-triazine), ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) were purchased from Sigma-Aldrich (Steinheim, Germany).

The lipid peroxidation level was determined using the malondialdehyde (MDA) measurement method (Heath and Packer, 1968 (link)). Leaf samples (1 g FW) were homogenized in 10 ml of trichloroacetic acid (Sigma-Aldrich). The homogenate was centrifuged for 5 min at 15,000 × g. A 1-ml aliquot of the supernatant was mixed with 4 ml of thiobarbituric acid containing 20% (w/v) trichloroacetic acid (Sigma-Aldrich). The mixture was heated at 95°C for 30 min before cooling quickly in an ice bath and then centrifuged at 10,000 × g for 10 min.
Using the spectrophotometer (Shimadzu Corporation), the absorbance of the supernatant was recorded at 532 and 600 nm. The MDA level was calculated by multiplying the absorbance difference (A532–A600) by the molar extinction coefficient (155 mM⁻¹ cm⁻¹) and the results were expressed in nmol g^-1 FW.