Astaxanthin

Astaxanthin (Sigma-Aldrich) dissolved in dimethyl sulfoxide (DMSO) was stored in nitrogen at −80 °C, and the cells were treated with Astaxanthin (1, 2, or 5 µM) for 3 h, followed by stimulation with G (10 mM)/GO (5 mU/mL) for 30 min (to measure intracellular ROS levels), 8 h (to determine Ku levels, Ku–DNA binding activity, and Ku ubiquitination), and 16 h (to assess cell viability, apoptosis marker annexin V-stained cells, and DNA damage marker levels, i.e., γ-H2AX and DNA fragmentation).
To determine the mechanism of Ku degradation, the cells were pre-treated with MLN4924 and MG132 (dissolved in DMSO and diluted in RPMI 1640 to 5 nM and 0.5 µM, respectively) for 1 h, followed by stimulation with G/GO for 16 h. Control cells without Astaxanthin, MLN4924, or MG-132 treatment were incubated with less than 0.1% DMSO.

Liquid chromatography on an Acclaim UHPLC C-18 column (length 150 mm × 4.6 mm ID, 2.5 μm, Thermo Fisher Scientific, Bremen, Germany) as reported previously. The mobile phases were 1% formic aqueous solution (A), methanol 1% formic acid (B), and acetonitrile 1% formic acid (C). The gradient program time (min) (% B, C) was: (0.00, 18, 75); (5.00, 18, 75); (15.00, 40, 60); (20.00, 100); and 12 min for column equilibration. A curve was performed with astaxanthin (Sigma Aldrich, Santiago, Chile), covering six points, with standard solutions from 0.1 to 1 μg/mL, R² = 0.9999 (LOD: 3.22 LOQ 10.43 μg/L, standard recovery rate of astaxanthin: 99.51%). The flow rate was set at 1.00 mL min⁻¹, and the injection volume was 10 µL. Standards and extracts dissolved in methanol were kept at 10 °C in the autosampler. The detection wavelengths were 280, 330, 515 and 490 nm, and the diode array detector was recorded from 200 to 800 nm for peak characterization. For quantitative HPLC analysis, each sample was run in triplicate and the peak was monitored through DAD detector at 490 nm wavelength, similar to that previously reported for other extremophiles [17 (link),38 (link)].

The antioxidant activity of the extracts was analyzed using the Ferric-reducing antioxidant power (FRAP) assay. In this assay, Fe³⁺ is reduced to Fe²⁺ by the antioxidant compounds and forms a colored complex with 2,4,6-tripyridyl-s-triazine in acetate buffer (pH 3.6) at 593 nm [23 (link)]. For the assay, the reactive solution was freshly prepared with 25 mL of 300 mM acetate buffer, pH 3.6, and 2.5 mL of 20 mM FeCl₃·6H₂O in deionized water, as well as 2.5 mL of 10 mM 2,4,6-tripyridyl-s-triazine in 40 mM HCl. The FRAP working solution (300 µL) was mixed with the acetonic algae extract (30 µL), and after 30 min, the absorbance was recorded by a SPECTROstar Nano absorbance microplate reader (BMG LABTECH, Ortenberg, Germany). The calibration was performed with astaxanthin (Sigma Aldrich, St. Lewis, MO, USA) in acetone, and the results are expressed in astaxanthin equivalent μmol_Ax·g⁻¹ lyophilized biomass.