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Immunohistochemistry Immunohistochemistry and IHC scoring systems were performed as described previously 14�C17. The antibodies were anti-ANGPTL3 (Santa Cruz Biotechnology). To calculate the 5-year survival rate, we surveyed each patient's life and month of death. Transfection with shRNA plasmid Stable transfection was performed using Lipofectamine LTX and thiram Plus Reagents (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions. ANGPTL3 shRNA (shANGPTL3) and the control shRNA (shMock) (Santa Cruz) vectors were transfected into HSC-3 and Sa3. After transfection, the cells were isolated by the culture medium containing 1?mg/mL Puromycin (Invitrogen). After 3�C4?weeks, resistant cell colonies were picked and transferred to new dishes. ShANGPTL3- and shMock-transfected cells were used for further experiments. Cellular growth Cellular growth assay was performed as described previously 14�C17. Cell-cycle analysis In order to synchronize cells at the G0/G1 or G2/M transition, they were deprived of serum for 48?h or treated with 200?ng/mL nocodazole (Sigma, St. Louis, MO, USA) for 20?h according to the previous reports 18,19. Cell-cycle analysis was performed as described previously 14�C17. Tumorigenesis and tumoral growth in vivo To investigate whether ANGPTL3 expression contributed to tumorigenesis and tumoral growth, we used xenograft models in two cell lines (HSC-3 and Sa3). The cells (2?��?107) were independently injected selleck products subcutaneously into the backs of female nude mice, BALB/c-nu, purchased from Oriental Yeast Co. (Tokyo, Japan). All experimental animals were treated and cared for in accordance with institutional guidelines. The tumoral Erastin mouse sizes were measured using a digital caliper every 3�C4?days after injection, when the volume of the transplantation tumor reached 100?mm3. We used the formula 4��/3?��?(width/2)2?��?(length/2) to calculate tumoral volume. The mice were sacrificed after 28?days. Tumor tissues were fixed in 10% formalin, and paraffin sections (4?��m) were prepared for hematoxylin and eosin staining and IHC of anti-ANGPTL3, anti-ERK1/2, anti-pERK1/2, and anti-Ki-67 (Santa Cruz Biotechnology). Statistical analysis In comparisons of ANGPTL3 expression levels, statistical significance was evaluated using the Mann�CWhitney U-test. Relationships between ANGPTL3 IHC staining scores and clinicopathological profiles were evaluated using the ��2 test, Fisher's exact test, and Mann�CWhitney U-test. Survival curves were obtained using the Kaplan�CMeier method, and differences in survival rates between ANGPTL3-positive and -negative cases were compared using the log-rank test. P?
