Figure 4.

Validation of DNA microarray data and immunohistochemical analysis. (A) Validation of DNA microarray data by real-time PCR. The up-regulation (HMOX1, TGFBI, LY6D, and S100P) and down-regulation (EIF4EBP2) of genes associated with annexin A11 expression (ANXA11) and the dynamic response of gene expression to cisplatin treatment (DHRS2 and PCSK9) were validated using real-time PCR. N represents control cells and R represents RNAi cells. For each individual gene, the expression levels at different time points were normalized to the control sample (N, PCR, 0 h). In addition, the relative mRNA expression levels were normalized to GAPDH expression. Each gene was amplified in triplicate, and each experiment was performed three times. *P < .05, R versus N. **P < .05, either [R2 (or R3 or R4) vs R1] or [N2 (or N3 or N4) vs N1]. (B) Suppression of annexin A11 upregulated HMOX1 and LY6D protein expressions. Immunoblot analysis was performed to confirm the suppression of annexin A11 protein expressions in the cells. The up-regulations of HMOX1 and LY6D protein expression levels in R1, R2, R3, and R4 compared with N1, N2, N3, and N4 were demonstrated. β-Actin was taken as an additional control for equal sampling in immunoblot analysis. (C) Annexin A11 immunointensity inversely correlated with HMOX1 immunoreactivity in ovarian cancer patients. Two representative pairs of tissue sections (left two sections from a primary tumor with low EDR and right two sections from a first recurrent tumor with extreme EDR) stained with two different antibodies are shown. Both sections of each pair were from similar areas of the same specimen. Original magnifications: upper panel, x100; lower panel, x400.