Ruthenium red rur

The following reagents were used: 2-[5-[[(2,6-Dichlorophenyl)methyl]thio]-1,3,4-thiadiazol-2-yl]-pyrazine (Yoda1, Tocris Bioscience, Bristol, UK), GSK1016790 A (GSK, Sigma/Aldrich, St. Louis, MO, USA), HC-067047 (HC, Sigma/Aldrich), GdCl₃ (Gd³⁺, Sigma/Aldrich), and ruthenium red (RuR, Sigma/Aldrich). Each reagent was dissolved in the vehicle recommended by the manufacturer.

Proliferating and/or differentiating cells were treated with 5 μM phorbol 12-myristate 13-acetate (PMA), 200 μM ascorbic acid (AA), 5 mM N-acetyl-L-cysteine (NAC), 1 mM hydrogen peroxide (H₂O₂), 15 mM lithium chloride (LiCl) or 0.5 μM ruthenium red (RuR) (all from Sigma-Aldrich). Different time ranges for cell treatments were applied. H₂O₂ or PMA were used as positive controls for redox state and ROS detections, and were added to untreated proliferating cells for 1 h or 24 h, respectively. AA or NAC were added to untreated proliferating cells for 24 h. Then, the cell differentiation was initiated and the AA-pre-treated- and NAC-pre-treated cells were respectively exposed to AA or NAC for two periods: a long period that corresponds to a drug exposure along the 72 h of differentiation (i.e. full treatment); a shorter one that consists in a drug exposure for the first 24 h of differentiation followed by an incubation of the cells in drug-free differentiating medium for the next 48 h (i.e. short treatment). LiCl or RuR were added to untreated proliferating cells for 1 h. Then a further exposure of the cells to LiCl for 24 h or to RuR for 2 h was performed at the onset of the differentiation process, followed by an incubation of the cells in drug-free medium up to the third day of differentiation. RuR effect was also examined for a full treatment along the 72 h of differentiation.

GdCl₃ (Sinopharm Chemical Reagent Company, Beijing, China), ruthenium red (RuR) (Sigma-Aldrich, St. Louis, MO, USA), 6-N,N-Diethyl-β-γ-dibromomethylene-D-adenosine-5′-triphosphatetrisodium salthydrate (ARL67156) (Sigma-Aldrich, St. Louis, MO, USA) and apyrase (Sigma-Aldrich, St. Louis, MO, USA) were prepared with distilled water. 2-Methyl-1-[3-(4-morpholinyl)propyl]-5-phenyl-N-[3-(trifluoromethyl)phenyl]-1H-pyrrole-3-carboxamide (HC067047) (Tocris, Minneapolis, MN, USA) was dissolved in dimethyl sulfoxide (DMSO). All of these stock solutions were kept at −20 °C and diluted into working solutions to final concentrations when used. DMSO was kept at less than 0.1% in the final solution. Finally, 4% paraformaldehyde (PFA) (Sinopharm Chemical Reagent Company, Beijing, China) was prepared in a fume hood and stored at 4 °C.