1260 infinity 2 lc system

CIP3 was designed based on rational design as previously described.⁷⁰ (link)^,71 (link)^,72 (link)^,73 (link) Peptides were chemically synthesized using a fully automated peptide synthesizer (Syro I, Biotage) on solid support by following the solid-phase peptide synthesis (SPPS) methodology⁷⁴ using the fluorenyl-methoxycarbonyl (Fmoc)/tert-butyl (tBu) protocol. Final cleavage and side-chain deprotection were done manually. The peptides were analyzed using analytical reverse-phase high-pressure liquid chromatography (RP-HPLC) (1260 Infinity II LC System, Agilent, CA, USA) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) (autoflex® maX, Bruker, Billerica, MA, USA), and purified by preparative RP-HPLC (1260 Infinity II LC System, Agilent, CA, USA). The full description of peptide synthesis is provided in the supplementary information. In yeast experiments, the peptide was added to G1- arrested cells for 1 hour before they were released into the cell cycle.⁷⁵ (link)^,76 (link) A detailed protocol for peptide synthesis and purification will be sent upon request.

Doxorubicin and fluorescein concentrations were determined using fluorescence. All HPLC analyses were performed on a 1260 Infinity II LC System from Agilent Technologies (Santa Clara, CA) equipped with an XBridge Oligonucleotide BEH C18 column from Waters (Milford, MA). The temperature was set at 40 °C, and the injection volume was 22.5 µL. TEAA mobile phase (100 mM triethylamine/acetic acid, pH = 7.00) (Fisher Scientific, Waltham MA) was used for ion-pairing binding to the column, and 100% HPLC grade acetonitrile was used for elution. The UV detector was set to measure absorbances at 260 and 480 nm. The fluorescence detector was set with an excitation wavelength of 480 nm and multiple emissions with wavelengths of 520, 550, 595, and 650 nm and a PMT gain of 18. To determine the doxorubicin or fluorescein fluorescence from each chromatogram, the AUC was measured with the software Chem Station provided by Agilent Technologies (Santa Clara, CA). A standard curve of doxorubicin and fluorescing fluorescence was obtained before sample analysis. Samples were run in triplicate. Fluorescence AUC was converted to concentration with the standard curve and plotted through time (for serum or plasma samples, standard curves were acquired with serum or plasma spiked with the relevant analyte).

Papaya extracts were separated in a Hydro-RP C-18 column (250.0 mm × 4.6 mm, 5 µm, Phenomenex, Torrance, CA, USA) equipped with HPLC system (1260 Infinity II LC system, Agilent, Santa Clara, CA, USA) fitted with a diode array detector set at 340 nm. Eluents were passed through the column at a flow rate of 1 mL/min using a gradient of solvent A (water) and solvent B (acetonitrile) in the following sequence: 0–5 min, 2% B; 5–12 min, 2–5% B; 12–17 min, 5–8%; 17–65 min, 8–30% B; 65–68 min, 30% B; 68–78 min, 30–50% B; 78–100 min, and 50–100% B. For the quantification of phenolics, relative retention times and relative peak areas were compared using fifteen compounds as standards: acacetin (CAS 480-44-4), apigenin (CAS 520-36-5), apigetrin (CAS 528-74-5), chlorogenic acid (CAS 327-97-9), cryptochlorogenic acid (CAS 905-99-7), cynarin (CAS 30964-13-7), diosmin (CAS 520-27-4), eridictol (CAS 20126-59-4), eupatorine (CAS 855-96-9), isoschaftoside (CAS 52012-29-0), linarin (CAS 480-36-4), luteolin (CAS 491-70-3), luteoside (CAS 5373-11-5), neochlorogenic acid (CAS 906-33-2), and vicenin II (CAS 23666-13-9).