Immunohistochemical staining of coronin-1C Immunohistochemistry (IHC) was performed on 4-5 μm thick paraffin sections. Sections were deparaffinized and rehydrated with graded ethanols. For immunostaining, VECTORSTAIN ABC kit (Vector Lab, CA) was used according to the manufacturer’s instructions. Primary antibodies used were rabbit anti-coronin-1C (Protein Tech Group, CA). Tumor development of spontaneous pulmonary selleck screening library metastasis in nude mice model of HCC Highly spontaneous metastatic nude mice
model (HCCLM9 group) of human HCC was used to study the relationship between coronin-1C levels and tumor BVD-523 supplier progressive and metastasis. Twenty-four nude mice (HCCLM9) were produced as described previously. The mice were randomly divided into three groups of eight mice in each group. At the end of the fourth, fifth and sixth wk, one group of was sacrificed. Liver cancer and lung samples were stored -80°C refrigerator. Clinical validation HCC specimens from 115 patients including 96(83.5%) males and 19(16.5%) females with mean age (M ± SD) of 47.9
± 12.4 years (range 18-78) were obtained from Fanpu Biotech, Inc. All tumors were fixed with formalin and embedded with paraffin. Ten high power field of each tissue section were selected randomly and observed double blind by two investigators. The staining score of each section were calculated by staining intensity and positive rate of cancer cells. Staining intensity: the score of 3-deazaneplanocin A no staining, weakly staining and strong staining is 0, 1 and 2 respectively. Positive rate of cancer cells: 0-20% was recorded as 0; 20-50% was recorded as 1; >50% was recorded as 2. The sum of scores was computed as the score of staining intensity added the score of the positive rate of cancer cells. Then it was graded according the sum of scores: 0 (-); 1-2 (+); 3-4 (++). Statistical analysis All the experiment data were integrated into a comprehensive data set. Numerical data were recorded Ponatinib molecular weight directly. Chi-square test and Fisher’s exact test were used to compare the clinicopathologic parameters among patients with different
level of coronin-1C expression. Statistical analysis was performed on SPSS software version 13.0 (SPSS Inc. Chicago, IL), and P < 0.05 was considered as statistically significant. Results Differential expression of coronin-1C between HCCLM9 and MHCC97L cell strains as identified by ESI-MS/MS Membrane proteins were extracted from MHCC97L and HCCLM9 HCC cells and compared by SDS-PAGE analyses [Fig. 1A]. The differential and interesting protein bands were excised and analyzed by ESI-MS/MS. A total of 14 proteins were identified by ESI-MS/MS among the differential bands [Table 1, Fig. 1A]. Coronin-1C, a promising candidate, was identified with high confidence [Fig. 1B]. Figure 1 Coronin-1C was identified as differentially expressed protein between HCCLM9 and MHCC97L cells.