Cis diammineplatinum 2 dichloride

Cisplatin [cis-diammineplatinum(II) dichloride] (Sigma-Aldrich) was dissolved in 0.150 M NaCl. Aliquots were stored at −20°C for up to a maximum of three months, and thawed immediately before use.
Cells (1×10⁴) were seeded in 96-well plates and allowed to adhere overnight at 37°C. Briefly, following treatments of cells with Cisplatin (0; 1; 5; 7.5; 10; 25; 50; 100 µM) for 48 h, MTT reagent [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was added to each well, and incubated for 4 h at 37°C, in the dark. The media were removed, 100 µl of 1 N HCl/isopropanol (1∶25) was added in each well, and incubated for 15 min at room temperature under gentle agitation. Finally, absorbance was measured at 595 nm. Three independent experiments were performed in triplicates.

cis-Diammineplatinum (II) dichloride (product number: 479306, purity ≥ 99.9%), curcumin from Curcuma longa (product number: c1386, purity ≥ 65%), α-tocopherol (product number: 258024, purity ≥ 95.5%), Bradford assay kits and 10% neutral buffered formalin solution were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA). Thiobarbituric Acid Reactive Substances (TBARS), SOD and catalase assay kits were obtained from Cell Biolabs, Inc (San Diego, CA, USA). RNeasy mini kit, Omniscript RT kit and HotStar Taq DNA polymerase were obtained from Qiagen (Hilden, Germany). All other chemicals were of analytical grade.

Human non-small cell lung cancer (NSCLC) cell lines, A549 and H460 and monocytic cell line, THP-1 were purchased from the American Tissue Culture Collection (ATCC). Cells are maintained under the conditions recommended by the vendor. Cisplatin-resistant A549 and H460 cells were generated according to an established protocol [8 (link)] where a continued exposure to escalating concentration of cisplatin (CDDP) over the period of 6 months. Cisplatin [cis-diammineplatinum (II) dichloride] was purchased from Sigma-Aldrich and made into 0.20 M stock solution in NaCl and aliquoted. All cells were acquired or purchased with confirmations for the genotypic authentications from suppliers, and regularly tested for mycoplasma contamination.

cis-Diammine platinum (II) dichloride, nitro blue tetrazolium, N-(1-naphthyl) ethylenediamine, and Tris-HCl were purchased from Sigma (St. Louis, MO, USA). Thiobarbituric acid and trichloroacetic acid were purchased from Merck. All other chemicals and reagents used in this study were of analytical grade. Double-distilled water was used as the solvent.

A549, MCF-7 and MDA-MB-231 cells were obtained from American Type Culture Collection and cultured according to its instructions. All media were supplemented with 10% fetal bovine serum (FBS). The cell culture media and reagents were purchased from Sigma-Aldrich (Sigma-Aldrich, Milan, Italy). Doxorubicin hydrochloride (Sigma-Aldrich, Milan, Italy) was dissolved in sterile water. Cis-diammineplatinum (II) dichloride (Sigma-Aldrich, Milan, Italy) was dissolved in PBS. Tetramethylrhodamine-ethyl-ester (TMRE) (Thermo Fisher Scientific, MA, USA) was dissolved in dimethylsulfoxide.
Trabectedin was supplied by PharmaMar (Colmenar Viejo, Madrid, Spain), dissolved in dimethylsulfoxide to 1mM, and stored at -20°C. Cells were treated with trabectedin as indicated in Figure Legends, extensively washed, and incubated under standard conditions until analyzed.

Polyphosphates with chain lengths of 14, 60, 130, and 300 P_i were kindly provided by T. Shiba (RegeneTiss, Japan) and aliquoted to avoid repeated freeze-thaw cycles. A cis-Diammineplatinum(II) dichloride (Sigma-Aldrich) stock solution (3 mM) was prepared in double distilled water. The plasmid pETM41-EcPPXc, which encodes the Maltose Binding Protein (MBP)-EcPPXc was a kind gift from Florian Freimoser (Addgene plasmid #38329; http://n2t.net/addgene:38329; RRID:Addgene_38329) (22 (link)). To generate the mCherry-EcPPXc fusion protein, EcPPXc was cloned into the mCherry-containing pTEV5 vector between BamHI and NotI sites. To generate the alternative GFP-EcPPXc probe, GFP was amplified from the pEGFP-N2 vector with flanking regions containing NdeI and BamHI recognition sequences, and inserted to replace mCherry in the mCherry-EcPPXc construct. The purification of His-tagged mCherry-EcPPXc, GFP-EcPPXc, and their respective His-tagged control protein mCherry and GFP was done using a Ni-NTA column (Qiagen, Hilden, Germany). All reagents were purchased from Thermo Fisher Scientific (Waltham, MA, USA), Sigma-Aldrich (St. Louis, MO, USA) or New England Biolabs (Ipswich, MA, USA) unless specified otherwise.