WT cells (transfected with CXCR4 siRNAI, II/dextran-spermine, 150 ng/g body weight) through the tail vein. In group C, the animals were given IV injections of 1 �� 105 CT26.WT cells (transfected with naked CXCR4 siRNAI, II, 150 ng/g body weight) through the tail vein. In group inhibitor Regorafenib D, the animals were given IV injections of 1 �� 105 CT26.WT cells (transfected with naked CXCR4 siRNAI, II, 150 ng/g body weight) through the tail vein. Postinjection of naked CXCR4 siRNAI, II was done twice weekly (150 ng/g body weight) through the tail vein. In group E, the animals were given IV injections of 1 �� 105 CT26.WT cells (transfected with CXCR4 siRNAI, II/dextran-spermine, 150 ng/g body weight) through the tail vein. Postinjection of CXCR4 siR-NAI, II/dextran-spermine was done twice weekly (150 ng/g body weight) through the tail vein.

In group F, the animals in group F were given nonspecific control siRNA duplexes twice weekly via injection through the tail vein without any injection of CT26.WT cells. Animals were sacrificed after 35 days. RNA preparation and real-time reverse transcription-polymerase chain reaction analyses Total RNA was isolated from six groups of mice frozen liver tissues (50 mg) using Trizol reagent, according to the manufacturer��s instruction. Real-time quantitative RT-PCR was performed in the real-time PCR machine (tubes) RotorGene 3000 (Corbett) with Quantitect SYBR green RT-PCR one-step kit as described by the manufacturer. Briefly, 500 ng of RNA was placed into a 20 ��L reaction volume containing 0.2 ��M of each primer, 10 ��L of SYBER green I RT-PCR one-step master mix, and 0.

2 ��L of reverse transcriptase. A typical protocol included reverse transcription at 50��C for 30 minutes and a denaturation step at 95��C for 15 minutes, followed by 40 cycles with 94��C denaturation for 15 seconds, 57��C annealing for 30 seconds, and 72��C extension for 30 seconds. Melt curve analysis: melt data acquiring to Cycling A (FAM/Sybr), ramp from 72��C to 95��C, hold 45 seconds on the first step, hold 5 seconds on the next step. Two equations was used to calculate relative changes in CXCR4 and ��-actin expression from real-time quantitative RT-PCR experiment.

25 Ratio?=???(Etarget)��Cttarget(Ereference)��Ctreference (1) whereas ��Cttarget = Ctcontrol �C Cttreatment; ��Ctreference = Ctcontrol �C Cttreatment Ratio?=??2-����Ct (2) whereas ����Ct = ��Ctreference �C ��Cttarget The one-way analysis of variance and least significant difference post hoc test were used to determine whether there were any significant differences among the means of CXCR4 expression of groups A, B, C, D, Carfilzomib and E. Serum alkaline phosphatase measurement Serum ALP of animal serum was measured in animals by Hitachi (902 Automatic Analyzer, Berlin, Germany) after 35 days. The blood samples were obtained by heart puncture and were allowed to clot.