Ooled on ice and 4-micron sections were reduce 606143-89-9 web applying a Microm HM 310 microtome. Paraffin embedded sections had been floated inside a Thermo Scientific Tissue Flotation bath filled with water heated to 44uC before mounting on pre-cleaned, positively-charged glass slides. Slides were placed upright and allowed enough time to air dry. Hematoxylin and eosin 181223-80-3 staining H&E staining was performed working with Thermo Scientific 
Shandon Varistain Gemini automated stainer. The Gemini stainer was programmed as follows. Slides had been deparaffinized in the heater block for 20 minutes. The program then continued with incubation of slides in 3 changes of Q-Score method for quantification of nuclear immunohistochemical staining Q-score is a semi-quantitative method of tissue scoring employing the formula Q = P x I, where P is equal to the percentage of positively staining cells and I is equal to the intensity at which each positve cell stains. I or intensity, is given a value between 1 and 3, representing Mitochondria-Targeted Drugs low, medium or high staining intensity. To score Ki-67 expression in control verses TP187 treated tumor sections, three tumor sections from each treatment were examined. For each, at least 10 random fields had been photographed and P and I values have been calculated for each individual field. The average Q-score and the corresponding SEM have been calculated in this way for each treatment. Statistical analysis Statistical analysis was performed for tumor volumes and Qscoring of immunohistochemical staining in Microsoft Excel using the Student’s t-test, assuming unequal variances. P-values less than 0.05, obtained by this method were considered to be significant. Flow cytometric analysis of TP421 uptake The fluorescent properties of TP421 had been exploited to investigate the cellular uptake of TP compounds. PC3 prostate cancer cell lines have been seeded at a density of 5.06105 cells/dish in 33 mm tissue culture-treated dishes and allowed to adhere overnight in 2 mL growth media. The following day cells were collected by trypsinization, washed and resuspended in 500 mL 1x PBS. Three minutes prior to, and immediately following addition of 10 mM TP421 or DMSO, as vehicle control, fluorescence versus time was recorded for emission wavelengths between 425475 nM in response to excitation with the 355 nM UV solid state laser of the BD LSR II flow cytometer. media to each well. The plate was subsequently incubated overnight at 37uC in the presence of 5% CO2. At the onset of bioenergetics measurements, the assay medium was warmed to 37uC, and the pH was adjusted to 7.4. Culture media was removed from the XF24 cell microplate, and the wells have been rinsed with 1 mL assay medium. Next, 600 mL of the assay medium was added to each well, and the XF24 cell microplate was incubated at 37uC for 1 h without CO2 supplementation. Dilutions of the stock compounds have been prepared in assay media. A volume of 60 mL reagent was added the injection PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19888037 ports of the biosensor cartridge, and had been maintained at 37uC without CO2 supplementation. Calibration and assay measurements were performed at 37uC. The data generated reflects measurements taken over a treatment period of seven hours. Bioenergetics assay medium The bioenergetics assay medium was prepared by dissolving DMEM base in 500 mL distilled water. 1.85 g of sodium chloride was dissolved separately in 500 mL of distilled water. Solutions of sodium chloride and DMEM base were then combined and 20 mL of this combined solution was r.Ooled on ice and 4-micron sections had been reduce using a Microm HM 310 microtome. Paraffin embedded sections had been floated in a Thermo Scientific Tissue Flotation bath filled with water heated to 44uC before mounting on pre-cleaned, positively-charged glass slides. Slides have been placed upright and allowed enough time to air dry. Hematoxylin and eosin staining H&E staining was performed using Thermo Scientific Shandon Varistain Gemini 
automated stainer. The Gemini stainer was programmed as follows. Slides had been deparaffinized in the heater block for 20 minutes. The program then continued with incubation of slides in 3 changes of Q-Score method for quantification of nuclear immunohistochemical staining Q-score is a semi-quantitative method of tissue scoring using the formula Q = P x I, where P is equal to the percentage of positively staining cells and I is equal to the intensity at which each positve cell stains. I or intensity, is given a value between 1 and 3, representing Mitochondria-Targeted Drugs low, medium or high staining intensity. To score Ki-67 expression in control verses TP187 treated tumor sections, three tumor sections from each treatment had been examined. For each, at least 10 random fields were photographed and P and I values had been calculated for each individual field. The average Q-score and the corresponding SEM had been calculated in this way for each treatment. Statistical analysis Statistical analysis was performed for tumor volumes and Qscoring of immunohistochemical staining in Microsoft Excel making use of the Student’s t-test, assuming unequal variances. P-values less than 0.05, obtained by this method have been considered to be significant. Flow cytometric analysis of TP421 uptake The fluorescent properties of TP421 were exploited to investigate the cellular uptake of TP compounds. PC3 prostate cancer cell lines were seeded at a density of 5.06105 cells/dish in 33 mm tissue culture-treated dishes and allowed to adhere overnight in 2 mL growth media. The following day cells had been collected by trypsinization, washed and resuspended in 500 mL 1x PBS. Three minutes before, and immediately following addition of 10 mM TP421 or DMSO, as vehicle control, fluorescence versus time was recorded for emission wavelengths between 425475 nM in response to excitation with the 355 nM UV solid state laser of the BD LSR II flow cytometer. media to each well. The plate was subsequently incubated overnight at 37uC in the presence of 5% CO2. At the onset of bioenergetics measurements, the assay medium was warmed to 37uC, and the pH was adjusted to 7.4. Culture media was removed from the XF24 cell microplate, and the wells have been rinsed with 1 mL assay medium. Next, 600 mL of the assay medium was added to each well, and the XF24 cell microplate was incubated at 37uC for 1 h without CO2 supplementation. Dilutions of the stock compounds had been prepared in assay media. A volume of 60 mL reagent was added the injection PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19888037 ports of the biosensor cartridge, and had been maintained at 37uC without CO2 supplementation. Calibration and assay measurements were performed at 37uC. The data generated reflects measurements taken over a treatment period of seven hours. Bioenergetics assay medium The bioenergetics assay medium was prepared by dissolving DMEM base in 500 mL distilled water. 1.85 g of sodium chloride was dissolved separately in 500 mL of distilled water. Solutions of sodium chloride and DMEM base had been then combined and 20 mL of this combined solution was r.