The lysate was boiled Silodosin for 10 min and centrifuged at 10,000 g for 15 min. The protein samples were fractionated in 12.5% SDS polyacrylamide gel. The proteins on the gel were transferred to a PVDF membrane. After blocking with 10% nonfat milk, the membranes were washed with PBS containing 0.1% Tween 20, and then incubated with primary antibodies for 2 h at room temperature. After washing with PBS T, the membrane was incubated with secondary antibodies conjugated with horseradish peroxidase for 1 h. Finally, the signals on the membrane were detected by enhanced chemiluminescence detection reagents. In vivo Study Male nude mice were provided by the Laboratory Animal Services Center, The Chinese University of Hong Kong. HepG2 cells were subcutaneously inoculated into the back of each nude mouse.
After the tumor sizes had reached 80 mm 3, the tumor bearing nude mice were divided randomly into 3 groups: a control group, a low dosage IM group, and a high dosage IM group, with 7 mice in each group. IM was orally administered to mice every day for 14 consecutive days. The tumor size and body weight of each mouse were measured every 2 days during JAK Inhibitors the treatment period. After treatment, all mice were sacrificed. The tumors were dissected and the toxicity of IM towards the heart and liver was assessed by measuring the activities of creatine kinase, lactate dehydrogenase, alanine Sorafenib 475207-59-1 transaminase and aspartate transaminase in the plasma using assay kits purchased from Stanbio Co. Ltd. Tumor volume was calculated using the following formula: ! length ! width ! height.
were incubated with various concentrations of IM MEK Signaling Pathway for 48 h. The IC 50 value for each cell line is indicated in table 1. Among the different cell lines, HepG2 cells showed the lowest IC 50 value, i.e. the highest sensitivity to IM. Thus, HepG2 was selected as the target for further studies. As shown in figure 1 a, IM significantly inhibited the growth of HepG2 cells in a dose and time dependent manner. The IC 50 values were 101.2, 60.5, and 22.4 M for 24, 48, and 72 h, respectively. The cytotoxic effect of IM on normal liver cells was lower than that on HepG2 cells at a high concentration range. IM Induced Apoptosis on HepG2 Cells To determine if the antiproliferative effect of IM was associated with apoptosis induction, detection of apoptotic cells by fluorescence staining was performed.
Treatment of HepG2 cells with 30 or 60 M of IM resulted in an increased number of apoptotic cells when compared to the control. Genomic DNA cleavage is another important part of apoptosis. As shown in figure 2 b, a dose dependent occurrence of cellular ladder like DNA fragmentation was observed in HepG2 cells after 48 h of treatment imperial with IM. Quantification of apoptotic cells induced by IM was performed by annexin V binding assay. The externalization of PS from the inner leaflet of the plasma membrane to the cell surface is one of the hallmarks of early apoptotic cells. As shown in figure 2 c, IM induced apoptosis in HepG2 cells in a time and dosedependent manner. The percentage of apoptotic cells upon treatment with 0, 60, 120, and 180 M of IM was 3.7, Caspases play critical roles in apoptosis as the mediators of apoptotic signals from upstream molecules.