DN frgmenttion Effects of MC on poptosis of tumor ce were exm ined with FCSort flow cytometer Becton Dickinson using nnexin VFITCpropidum iodide PI; BD Phmin gen doube stining s previousy described . Fur thermore, observtions of poptotic bodies chrom tin coenstion were mde with Hoechst stining m mo visuized uer UV iumintion with NIKON TE microscope Nikon;  Moreover, poptotic bodies were counted in different fieds of Ridaforolimus microscopic observtion. One hured ces were exm ined in one fied. In ddition, DN frgmenttion ws determined with termin deoxynuceotidy trnsferse medited dUTP nick e being TUNE stining green fluorescence, using n In Situ Ce Deth Detec tion Kit Roche. Briefly, tumor ces were treted with MC  mo for hours, foowed by TUNE stining ccording to the mnufcturer instructions, monitored by flow cytometry. ssys of cecyce nysis mitochori deporiztion The cecyce distribution of ces fter MC tretment m mo, hours ws studied by using flow cytometry with PI stining m gm PI in PBS, contining Triton X m gm RNse ;

On the other h, mesurement of chnges of mitochori trnsmembrne potenti D É m ws mde using the sme procedure, but with JC stining t concentrtion of  gm . The mechnism is tht JC exists in mono meric form in the cytoso emitting green fluorescence so ccumutes s ggregtes in the mitochori emitting red fluorescence in norm ces. But in popto ticnecrotic ces, the D É m copse hts the mitochori ccumution of JC but mintins it in cytoso emitting green fluorescence;  Western bot nysis Effects of MC tretment on the expression Fingolimod phosphorytion of MPKs, b the ctivtion of poptotic cscdes, c the expression of Bc fmiy proteins were determined by Western bot . Briefly, ceur ystes were heted t C for minutes in  SDS oding buffer, foowed by SDS. The proteins were trnsferred to poyvinyidene difluoride membrne, which ws then incubted with primry ntibody in mik, foowed by incubtion with horserdish per oxidseconjugted ntimouse or ntirbbit secory ntibody visuized using the EC detection system mershm ife Science. Detection of nitric oxide production First, ces were incubted with m mo MC, b coincubted with m mo MC m mo NME nitric oxide NO synthse inhibitor, c coincubted with m mo MC m mo  p MPK inhibitor, respectivey, for hours.

NME or ws dded hour prior to MC. Seco, the genertion of nitrite nitrte, s the surrogte mrkers for NO, in the ce cuture superntnt ws determined by using Griess regent, buy Bergenin previousy described nim studies The in vivo ntitumor ctivity of MC ws studied in thymic nude nunu mice foowing the procedure reported esewhere with modifictions   First, tot of  CNE ces were trypsinized, wshed with  PBS, injected subcutneousy into the right flnk of ech mouse ice were checked ech dy for xenogrfttumor deveopment. Once the respiratory therapists tumors were ppbe, mice were romy divided into groups mice per group. The MC group received diy intr peritone injection of g MCkg body weight gkgd. The contro group ws treted with  PBS. Tumor voume ws determined every dys with cipers using the foowing formu: tumor voume mm ¼ ength mm  width mm .

Body weight ws moni tored every dys s n iictor of toxicity, mice were euthnized when purchase Bergenin tumor size exceeded , mm . TUNE stining ssy Oneday , remining mice were scrificed, sections of tumor tissue in both groups were prepred. s mentioned bove, TUNE ssy ws crried out using n In Situ Ce Deth Detection Kit Roche s per mnufcturer’s instructions. The ces were visuized uer ight micro scope, the percentge of poptotic ces uer hpf  ws ccuted   crjourns Cncer Prev Res;