Materials and

Temsirolimus mouse methods Cell line The HER-2 overexpressing human ovarian cancer cells SK-OV-3 [21] were obtained from the Cell Bank of Shanghai Institutes for Biological Sciences (Shanghai, China). They were cultured in DMEM (Gibco, USA) supplemented with 10% FBS (Gibco, USA) in an incubator with 5% CO2 and saturated humidity at 37°C. MTT assay SK-OV-3 (5 × 103 per well) cells were seeded in 96-well plates and cultured overnight. Then, the medium was replaced with fresh DMEM or the same medium containing ChA21 (prepared as described in previous studies [16, 17]) at concentrations of 0.067, 0.2, 0.6, LY2603618 clinical trial 1.8, 5.4 μg/ml for 72 h, or the cells were treated with ChA21 at the concentration of 5.4 μg/ml for 24, 48, 72, 96 h, respectively. MTT (Sigma, USA) with 20 μl samples was added to each well and incubated for an additional 4 h. Then culture medium was discarded and 150 μl dimethyl sulfoxide (DMSO) was added. OD 570 nm was measured by a multi-well scanning spectrophotometer (Multiskan MK3, Finland). The inhibitory growth rate was calculated as follows: (1 – experimental OD value/control OD value) × 100%. Inhibition of ChA21 on SK-OV-3 nude mice xenografts BALB/c female nude mice (6 weeks old, 18.0 ± 2.0 g) were obtained from Shanghai selleck compound Laboratory Animal Center (SLAC, China). SK-OV-3 cells (5 × 106 per mouse) were subcutaneously inoculated into the left flank of the mice. Tumor-bearing mice in which the tumor volume reached about 50 mm3 were selected,

and randomized, injected with either sterile normal saline or ChA21(40 mg/kg) twice weekly via caudal vein (i.v) for 5 weeks. Tumor size was measured twice a week and converted to tumor volume (TV) as the following formula: TV (mm3) = (a × b2)/2, where a and b are the largest and smallest diameters (in millimeters), respectively. All animals were killed after giving ChA21 or sterile normal saline for 5 weeks, and the transplantation tumors

were removed, weighed and fixed for further study. The tumor inhibition ratio (TIR) was calculated as follows: (1 – experimental mean weight/control mean weight) × 100% [22]. Evaluation of potential adverse effects To evaluate DCLK1 the potential side effects or toxicity on mice during treatment of ChA21, gross measures such as weight loss, ruffling of fur, life span, behavior, and feeding were investigated. The tissue of heart, liver, spleen, lung, kidney, and brain were fixed in 10% neutral buffered formalin solution and embedded in paraffin, and then stained with H&E. Transmission electron microscopy SK-OV-3 cells treated with ChA21 (5.4 μg/ml) for 72 h, as well as 1 mm × 1 mm tumor tissues from nude mice, were fixed with glutaraldehyde and osmium tetroxide. After dehydration in a graded series of acetone and steeping in propyleneoxide, the samples were ultramicrotomed after embedded in Epon 812. The sections were stained with lead citrate, and examined by an electron microscope (JEM-1230, Japan). TUNEL staining of apoptotic cells SK-OV-3 cells (2.