Figure 5

Detection of over expressed CIZ1 spliced variants in cancer cell lines. A: Schematic representation of CIZ1 exon 8 to exon 13 and the primers (Ciz1-ex8F-Ciz1-ex13R) used for amplification. Schematics I-III corresponds to the 3 bands seen in Fig. 5B (upper panel) which were verified by sequencing. Schematic I represents full length exon 8-13, II shows partial deletion of exon 8 and III shows partial deletion of exon 8 and skipping of exon 9. Schematic IV indicates primers used to amplify the product shown in Fig. 5B (middle panel) and in Fig. 5C and D. B: Upper, RT-PCR using the primer pair Ciz1-ex8F and Ciz1-ex13 R. Middle, RT-PCR using variant specific primers (Ciz1-jex8-ex12F - Ciz1-jex13-ex14R). Bottom, actin primers were used for normalization. The panel includes three normal cell lines (HFL1, ASF and TIG) and three cancer cell lines TTC466, SKNMC and H727). Stars indicate sequenced bands. C: Validation of microarray result for exon 8-12 with SKNMC and TTC466. Histogram shows average quantitative PCR result for 3 replicates with SEM. Results were normalised to Actin levels and calibrated to pooled reference RNA (8 normal cell lines including, IMR90, WI38, HEK293, MRC5, HFL1, ASF-4-1, TIG-2M-30 and RPM1788), as used in the microarray experiment. D: Quantitative PCR with Ciz1 splice variant specific primers (Ciz1-jex8-ex12F and Ciz1-jex13-ex14R). The panel includes lung (SBC2, SBC5 and H727), cervix (Hela), and Urothelial cancer cell lines (RT112, RT4 and EJ). Normal cell lines were also included (HFL and TIG) as well as the reference poole RNA, which was used as calibrator. Results were normalized to GAPDH.