Cellular extracts (30 μg) were incubated in a 96-well microtitre plate with 10 μl Ac-DEVD-pNA (2 mM) for 6 h at 37°C. Then caspase-3 activity was quantified in the samples with a microplate spectrophotometer (NanoDrop 2000c, Thermo Fisher Scientific Inc., USA) by the absorbance at a wavelength of 405 nm. All experiments were performed in triplicate. Statistical analysis Statistical analysis was performed using the SPSS 13.0 software. The relationship between PKCε expression and the clinicopathologic features of RCC was assessed by the Fischer’s exact test. Continuous data are expressed as mean ± standard deviation. Statistical significance was analyzed
IWP-2 mouse by one-way analysis of variance (ANOVA) followed by Bonferroni’s post-hoc test, with values of P < 0.05 considered statistically significant. Results PKCε expression in renal tissues The expression of PKCε protein in 15 specimens of normal renal tissues and 128 specimens of RCC was detected by immunohistochemistry SAR302503 with an anti-PKCε
monoclonal antibody. PKCε expression was weak in normal renal tissues, but strong in both cytoplasm and nuclei of RCC cells (Figure 1). The level of PKCε overexpression was significantly STA-9090 higher in RCC than in normal tissues (63.3% vs. 26.7%, P = 0.006). When stratified by pathologic type, no significant difference was observed among clear cell, papillary, and chromophobe RCCs (62.0% vs. 60.0% and 80.0%, P = 0.517). PKCε overexpression showed no relationship with the sex and age of patients with clear cell RCC (both P > 0.05), but was related with higher T stage (P < 0.05) and higher Fuhrman grade (P < 0.01) (Table 1). Figure 1 Immunohistochemical staining of PKCε in tissue specimens. PKCε is overexpressed in click here both cytoplasm and nuclei of clear cell renal cell carcinoma
(RCC) cells (A). Primary antibody isotype control (B) and normal renal cells (C) show no or minimal staining. The original magnification was ×200 for left panels and ×400 for right panels. Table 1 PKCε overexpression in human clear cell renal cell carcinoma tissues Group Cases PKCε overexpression P value (-) (+) Sex Men 69 24 45 0.365 Women 39 17 22 Age ≤ 55 years 43 16 27 0.599 >55 years 65 21 44 T stage T1/T2 89 38 51 0.028 T3/T4 19 3 16 Fuhrman grade G1/G2 86 39 47 0.002 G3/G4 22 2 20 PKCε, protein kinase C epsilon. PKCε expression in renal cell cancer cell lines We detected the expression of PKCε in five RCC cell lines using Western blot. PKCε was expressed in all five RCC cell lines at various levels, with the maximum level in clear cell RCC cell line 769P (Figure 2A). Immunocytochemical staining showed that PKCε was mainly expressed in both cytoplasm and nuclei, sometimes on the membrane, of 769P cells (Figure 2B).