Milli q integral 3 system

Methanol (LC-grade), formic acid (for MS), ammonium formate (for MS) and thymidine (THY) were purchased from Sigma-Aldrich (Seelze, Germany). Zidovudine (ZDV) and zidovudine-d₃ (ZDV-d₃) were purchased from Toronto Research Chemicals (Toronto, Canada). Purified water was generated from a Merck Millipore Milli-Q-Integral 3-System (Darmstadt, Germany). Stock solutions of target analytes and surrogate standard solutions were prepared as listed in Table 1 and stored in the dark at −25 °C. Working solutions containing all analytes were prepared in methanol at 10, 1, 0.1 and 0.01 mg L⁻¹, for surrogate standards at 1 mg L⁻¹ and were stored at 4 °C.Table 1

Compounds, solvents and concentrations of stock solutions, retention times, MRM transitions (bold printed transitions are the most intense), declustering potential, collision energy, cell exit potential, dwell time, transition ratios and assignment of internal standards (IS).

Table 1

compound	stock solutions		LC/MS/MS-parameters
	solvent	concentration [g L−1]	retention time [min]	[M+H]+	MRM1	MRM2	DP	CE1 [eV]	CE2 [eV]	CXP1 [V]	CXP2 [V]	dwell time [ms]	Assignment of IS
THY	H2O	1.0	7.2	243	127	110	32	17	44	10	10	50	ZDV-d3
ZDV	H2O	1.0	11.2	268	127	110	34	17	45	10	10	50	ZDV-d3
internal standard
ZDV-d3	MeOH	0.1	11.1	271	130	113	31	15	43	10	10	50

Apigenin, Senescence Cells Histochemical Staining Kit, Griess reagent were purchased from Sigma Chemicals Co., USA. Dulbecco's modified Eagle's medium (DMEM) and Roswell Park Memorial Institute 1640 medium (RPMI-1640) supplemented with l-glutamine, fetal bovine serum (FBS), penicillin, streptomycin, Dulbecco's phosphate-buffered saline (D-PBS) and Hank's balanced salt solution (HBSS) were all procured from Gibco (Invitrogen), USA. JC-1 fluorescent dye was obtained from Life Technologies (Thermo Fisher Scientific Corporation, Carlsbad, CA, USA). Phospho-Rb (Ser780) Antibody, Bax Antibody, Bcl-2 Antibody, Anti-mouse IgG and Anti-rabbit IgG were procured from Cell Signaling Technology®, USA while Anti-p21WAF1 (Ab-1) was obtained from Calbiochem®, Darmstadt, Germany. Cyclin D1, Cyclin E, p53, p16 antibodies were procured from Santa Cruz Biotechnology, Inc., Dallas, USA while β-actin antibody was obtained from Sigma Chemicals Co., USA. JC-1 fluorescent dye was obtained from Life Technologies (Thermo Fisher Scientific Corporation, Carlsbad, CA, USA). All other chemicals used were of the highest analytical grade available. The chemicals were used as obtained without further purification. Milli-Q water obtained from Milli-Q Integral 3 system (Merck Millipore, Germany) was used for all experiments.