Supporting Information Dhar and Lippard 10.1073/pnas.0912276106 SI Text The complexes cis -[Pt(NH 3 ) 2 Cl 2 ] (1) and c , c , t - [Pt(NH 3 ) 2 Cl 2 (OH) 2 ] (2) were synthesized as described. Distilled water was purified by passage through a Millipore Milli-Q Biocel water purification system (18.2 M) containing a 0.22-m filter. Anti-cytochrome c (Ab-1) sheep polyclonal antibody was pro- cured from Calbiochem. Alexa Fluor 488-labeled secondary antibody donkey anti-(sheep IgG) was obtained from Invitrogen for cytochrome c detection. For AIF detection, we used a rabbit polyclonal IgG antibody from Santa Cruz Biotechnology, Inc. Alexa Fluor 546-labeled secondary antibody goat anti-(rabbit IgG) was purchased from Invitrogen. The detection of the cisplatin 1,2-d(GpG) intrastrand adduct was carried out using a monoclonal adduct-specific antibody R-C18 which was kindly provided by Ju ¨rgen Thomale (University of Duisburg-Essen). FITC-labeled secondary antibody rabbit anti-(rat Ig) was ob- tained from Invitrogen. Specific adhesion slides for immunoflu- oresecence were purchased from Squarix Biotechnology. JC-1 (5,5,6,6-tetrachloro-1,1,3,3 tetraethylbenzimidazolylcarbo- cyanine iodide) was obtained from Cayman Chemicals. 1 H, 13 C and 195 Pt NMR spectra were recorded on a Bruker AVANCE- 400 NMR spectrometer with a Spectro Spin superconducting magnet in the Massachusetts Institute of Technology Depart- ment of Chemistry Instrumentation Facility (MIT DCIF). Atomic absorption spectroscopic measurements were taken on a Perkin-Elmer AAnalyst 300 spectrometer. HRMS analysis was carried out on a Bruker Daltonics APEXIV 4.7 Tesla Fourier Transform Ion Cyclotron Resonance mass spectrometer in the MIT DCIF. Fluorescence imaging studies were performed with an Axiovert 200M inverted epifluorescence microscope (Zeiss) equipped with an EM-CCD digital camera C9100 (Hamamatsu). An X-Cite 120 metal-halide lamp (EXFO) was used as the light source. The microscope was operated with Volocity software (Improvision). Electrochemistry. Electrochemical measurements were made at 25 °C on a EG&G PAR Model 263 Potentiostat/Galvanostat with electrochemical analysis software 270 and a three-electrode set-up comprising a glassy carbon working electrode, platinum wire auxiliary electrode and a Ag/AgCl reference electrode. The electrochemical data were uncorrected for junction potentials. KCl was used as a supporting electrolyte. Cytotoxicity Studies. Cell lines and cell culture. Human testicular cancer NTera-2, human cervical cancer HeLa, human osteosar- coma U2OS, human breast adenocarcinoma MCF-7, and healthy normal fibroblast GM61869 cell lines were obtained from the ATCC. Human ovarian carcinoma cell lines A2780 and A2780/CP were kindly provided by Thomas Hamilton (Fox Chase Cancer Center, Jenkintown, PA). A549 lung carcinoma and normal lung MRC-5 cells were obtained from David E. Root (Whitehead Institute for Biomedical Research). Cells were grown at 37 °C in 5% CO 2 in DMEM supplemented with 10% FBS and 1% penicillin/ streptomycin. Cells were passed every 3–4 days and restarted from frozen stocks upon reaching pass number 20. MTT assay. The cytotoxic behavior of mitaplatin, DCA, and cisplatin was evaluated by using the MTT assay. Solutions of the platinum complexes were freshly prepared in sterile PBS before use and quantitated by atomic absorption spectroscopy. Cells were seeded on a 96-well plate in 100 L of DMEM and incubated for 24 h. The cells were then treated with mitaplatin, DCA, or cisplatin, separately at varying concentrations and incubated for 72 h at 37 °C. The cells were then treated with 20 L of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro- mide (MTT) (5 mg/mL in PBS) and incubated for 5 h. The medium was removed, the cells were lysed by adding 100 L DMSO, and the absorbance of the purple formazan was re- corded at 550 nm using a BioTek Synergy HT multidetection microplate plate reader. Each condition was repeated in tripli- cate in three independent experiments for each cell line. Fluorescence Imaging. Cell fixing solution. Paraformaldehyde (4.0 g) and NaOH (0.4 g) were dissolved in 100 mL distilled water. To this solution was added NaH 2 PO 4 (1.68 g) and the pH was adjusted to be between 7.5 and 8.0 by adding NaOH. Fluorescence sample mounting media. For sample mounting, a solu- tion containing 20 mM Tris (pH 8.0), 0.5% N-propyl gallate, and 70% glycerol was used. Detection of cisplatin 1,2-d(GpG) intrastrand adducts. Detection of the platinum 1,2-d(GpG) adducts was carried out by following a procedure recently reported by us using an antibody specific to this adduct (3). Briefly, NTera-2 cells were seeded in a six-well plate using DMEM and incubated overnight at 37 °C. Mitaplatin was added to a final concentration of 20 M and incubated at 37 °C. After 12 h, cells were trypsinized, washed with PBS, resuspended in HAES-sterile-PBS at a density of 1 10 6 per mL and placed onto a precoated slide (ImmunoSelect, Squarix) and air dried. Cell fixing was carried out at 20 °C in methanol for 45 min. Nuclear DNA was denatured by alkali (70 mM NaOH, 140 mM NaCl, and 40% methanol, vol/vol) treatment for 5 min at 0 °C, and cellular proteins were removed by a proteolytic procedure involving two steps. The cells were first digested with pepsin at 37 °C for 10 min and then with proteinase K at 37 °C for 5 min. After blocking with milk (1% in PBS; 30 min; 25 °C) the slides were incubated with anti-(Pt- DNA) Mabs (R-C18 0.1 mg/mL in milk) (4) overnight at 4 °C. After washing with PBS, immunostaining was performed by incubation with FITC-labeled goat anti-(rat Ig) antibody at 37 °C for 1 h. The nuclei of the cells were stained by using Hoechst (H33258) (250 g/L) and mounted using the mounting solution for imaging. Determination of platinum concentrations from cell extracts. A549 and MRC5 cells were grown in DMEM to 95% conf luence in 175-cm 2 flasks. These cells were then treated either with 10 M cisplatin or mitaplatin and subsequently incubated for 24 h at 37 °C. Cells were washed with PBS three times and released by trypsinization into PBS. Solutions containing cells were then centrifuged at 800 g for 10 min and the cell pellet obtained was resuspended in 100 L of ice-cold lysis buffer (1.0 mM DTT, 1.0 mM PMSF, 10 mM KCl, and 10 mM MgCl 2 , pH 7.5) for 15 min. This process was repeated and finally the pellets were resuspended in 40 L of ice-cold lysis buffer. The cell membranes were lysed by 10 strokes of a 28-gauge syringe. The resulting suspension was centrifuged at 11,000 g for 20 min and the cytosolic fraction of the cells was collected as supernatant. The pellet was resuspended in 40 L of extraction buffer (1.0 mM DTT, 1.0 mM PMSF, 1.5 mM MgCl 2 , 0.2 M EDTA, 0.42 M NaCl, and 25% glycerol, pH 7.9) and lysed with 10 strokes of a 28-gauge syringe. The lysate was shaken at 1,000 rpm for 45 min at 4 °C and then centrifuged at 20,000 g for 10 min at 4 °C. The supernatant was collected as the nuclear fraction. Platinum concentrations in all of the fractions were determined by AAS. The protein concentra- tion in each fraction was determined by using bicinchoninic acid (BCA) assay. Total platinum concentrations were expressed as nanograms of Pt per microgram protein. Dhar and Lippard www.pnas.org/cgi/content/short/0912276106 1 of 10