Gs 6r

Western blotting was performed as previously described^{22 (link), 23 (link), 46 (link)}. The lysates from the xenografts or cell lines with different treatments were homogenized in RIPA lysis buffer using a homogenizer. The supernatants were collected by centrifugation (12,000 rpm at 4 °C for 25 min; Beckman GS-6R). Protein was resolved by electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The membranes were probed with monoclonal HIF-1α (NB100–105, Novus biological, Littleton, CO, USA), Bax (2772 S, Cell signaling, Boston, MA, USA), Bcl-2 (3498S, Cell signaling), caspase-3 (9661S, Cell signaling), PARP (5625, Cell signaling), and PCNA (18197, Abcam, Cambridge, MA, USA), as well as p21 (Sc817, Santa Cruz, CA, USA), CDK4 (Sc70831, Santa Cruz), SP1 (ab27595, Abcam), HK2 (2772S, Cell signaling), PKM2 (4053S, Cell signaling), LDH-A (3582S, Cell signaling), and PDK1 (3820 S, Cell signaling). Following incubation with the primary antibody overnight at 4 °C, membranes were washed with Tris-buffered saline (pH 7.2) containing 0.05% Tween-20 and subsequently incubated with horse radish peroxidase (HRP)-conjugated anti-mouse (Sc-2005, Santa Cruz) or anti-rabbit (Sc-2357, Santa Cruz) secondary antibody for 1 h at room temperature. Bands of interest were analyzed using the ChemiDocTM Touch Imaging System (BIO-RAD, Hercules, CA, USA).

To isolate CSCs from the chorionic membrane, the membrane was incubated in 1 mg/mL type II collagenase (Worthington Biochemical Cporporation, Lakewood, NJ, USA) at 37 °C, followed by filtering in 100, 70, and 30 μm nylon strainers. The cell digest was centrifuged (Beckman Coulter GS-6R) at 1500 rpm to pellet the CSCs, and a cell count was obtained using a Cellometer (Nexcelom Bioscience, Lawrence, MA, USA). To isolate ASCs from the amniotic membrane, the amniotic membrane was first treated with 0.25% trypsin for 25 min to remove amniotic epithelial cells. After the membrane was washed twice with DPBS, ASC isolation was performed in the same way as the CSC isolation mentioned above. Freshly isolated, uncultured ASCs and CSCs were used for RNA-seq, and cells with passages below five were used for the entire in vitro study.

Blood was collected and centrifuged at 3000 rpm (Beckman GS-6R, Germany) at 4 °C for 30 min to separate the serum. Serum ALT and AST levels were measured using spectrophotometric diagnostic kits (Roche, Germany).

Two types of extraction media were used to isolate pigments from algal cells: 90 % acetone solution with respect to chlorophylls and carotenoids (Parsons et al. 1984 ) and extraction medium consisted of 0.25 M Trizma Base, hydrated 10 mM disodium EDTA (2H₂O), and 2 mg cm⁻³ lysozyme; the initial pH 9 was adjusted to final 5.5 (HCl)—in case of extraction phycobiliproteins from cyanobacteria cells (according to Steward and Farmer 1984 ). Chlorophylls and carotenoids were extracted by mechanical grinding and sonication (2 min, 20 kHz, Cole Parmer, 4710 Series) in the darkness conditions at 4 °C for 2 h. Procedure of isolation of phycobiliproteins from cells was based on combination of a gentle mechanical grinding and enzymatic (lysozyme) reaction in order to successfully disintegrate cell walls and improve pigment extraction efficiency in darkened room conditions. Filers were then incubated at 37 °C for 2 h in a dry block heat bath (Thermoleader, Uniequip) and after that kept in dark at 4 °C for 24 h. The extract was then centrifuged (20 min, 5 °C, 3210×g, Beckman, GS-6R) to remove the filters and cellular debris.
The clarified extracts were then subjected to the chromatographic analysis in case of chlorophylls and carotenoids and spectrofluorometric measurements in case of phycobilins.

Blood samples were collected from the participants by venous catheter into heparinized tubes before and immediately after the physical test. Five blood samples and urine were taken: Before supplementation (basal value) (T1), after supplementation (two weeks) (T2), after first physical exercise test (T3), after 24 h of rest (T4), after second physical exercise test (T5). One aliquot of blood was collected in tubes with an EDTA anticoagulant for hematological analysis. The remaining blood was immediately centrifuged at 1750 g for 10 min at 4 °C in a Beckman GS-6R refrigerated centrifuge (Beckman, Fullerton, CA, USA) to separate plasma from red blood cell pellets. Plasma samples were immediately frozen and stored at −80 °C until analysis.