Figure 2: KIAA1199 expression correlates with HPV status in cervical cancer-derived cells and with cervical intraepithelial neoplasia progression.

(a) KIAA1199, BCL-3 and p65 expression levels in HPV-positive and -negative cervical cancer-derived cell lines. Anti-KIAA1199, -BCL-3 and -p65 WBs using protein extracts from the indicated cell lines are shown. (b) E6 depletion in a HPV16-positive cervical carcinoma-derived cell line decreases KIAA1199 mRNA levels. CaSki cells were transfected with the indicated siRNAs (E6 siRNA pool) and the resulting cells were subjected to real-time PCR to assess E6 or KIAA1199 mRNA levels. The abundance of E6 and KIAA1199 transcripts in cells transfected with the control siRNA (‘GFP’) was set to 1 and their levels in cells transfected with the other siRNAs were relative to that after normalization with 18S rRNA. Data from three independent experiments (means±s.d.) are shown (Student’s t-test P-values: **<0.01). (c) Ectopic HPV16 E6 expression in a HPV16-negative cell line enhances KIAA1199 expression. A431 cells were transfected with a control or an E6-expressing construct and cell extracts from the resulting cells were subjected to real-time PCR or to WB analyses. The abundance of BCL-3 and KIAA1199 transcripts in cells transfected with the control expression plasmid (‘empty vector’) was set to 1 and their levels in E6-expressing cells were relative to that after normalization with β-actin. Data from two real-time PCR analyses performed in triplicates (means±s.d.) are shown (Student’s t-test P-values: **<0.01). (d) KIAA1199 immunostaining in normal exocervix, cervical intraepithelial neoplasia grade I (CIN I), grade II (CIN II), grade III (CIN III) and invasive squamous cell carcinoma (SCC) areas. Original magnifications: × 200. (e) Semi-quantitative evaluation of KIAA1199 expression in these biopsies (see Methods for details). Asterisks indicate statistically significant differences compared with normal exocervix (Student’s t-test P-values: ***<0.001; **<0.01). (f) KIAA1199 expression is significantly higher in (pre)neoplastic lesions transformed by HPV16 compared with lesions associated with other HPV types. Semi-quantitative evaluation of KIAA1199 expression in normal exocervix, cervical intraepithelial neoplasia grade I (CIN I), grade II (CIN II), grade III (CIN III) and invasive squamous cell carcinoma (SCC) areas. Asterisks indicate statistically significant differences between HPV16-positive cases and cases infected with other HPV types (Student’s t-test P-values: **<0.01; *<0.05).