Article Figures & Data
Figures
- Figure 1.
Aberrant transcripts in primary human tumor tissues. A, schematic showing the location of the probes to identify the wild-type and aberrant transcript (exon 11 skipped). B, different types of human cancer cDNA analyzed by real-time PCR analysis for wild-type and aberrant E-cadherin transcript (n = 9 for each tumor, n = 3 for matched organ-specific normal tissue). Black bars represent the percentage of tumor cDNAs with E-cadherin expression less than 50% of matched normal tissue; open bars represent the percentage of tumor cDNAs in which the percentage of aberrant transcript is at least 2-fold greater than the aberrant transcript in matched normal tissue. For the head and neck cancer group, the data show relative levels of total E-cadherin expression in 8 pairs of matched normal (N) and tumor tissues (T), relative to actin and adjusted to the matched normal control. C, head and neck cancer data from 8 matched groups is shown with wild type and percentage of exon 11–skipped transcript in each matched pair (log scale).
- Figure 2.
Analysis of the aberrant transcript. A, RT-PCR analysis of untreated UMSCC12 cells treated with emetine or emetine plus actinomycin-D and analyzed for expression of E-cadherin RNA. With the same cDNA preparation FRS2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified. B and C, UMSCC14 and UMSCC12 cells were treated with actinomycin-D, and transcript-specific PCR conducted at different time points. D, relative expression of E-cadherin in tert keratinocytes, NHOK, and head and neck cell lines relative to expression in tert-kert cells. E, percentage exon 11–skipped transcript calculated by the cycle difference between the 2 transcripts with transcript-specific real-time PCR of untreated (E) and emetine treated, (F) cells.
- Figure 3.
Minigene experiments show similar splicing patterns. A, schematic showing the minigene constructs with E-cadherin exon 10, 11, and 15. B, transient transfection and RT-PCR analysis (DNA gel) of UMSCC12 cells transfected with various minigene constructs. Primers bind to β−globin exon E1 and E3. Upper arrow indicates the correct expected size and the lower arrow indicates a transcript lacking exon 11. C, schematic for the location of probes for transcript-specific PCR. D, real-time PCR data showing the percentage aberrant transcript in tert-kert and head and neck cell lines (mean ± SD).
- Figure 4.
Role of splicing factors in aberrant splicing. A, schematic with predicted binding sites for splicing factors around E-cadherin exon 11. B, real-time PCR data for expression of SFRS1, 2, 5, and 6 splicing factors in cell lines relative to tert-kert cells. C, expression of splicing factors by real-time PCR in primary human head and neck tumors relative to matched normal tissue (7 matched pairs).
- Figure 5.
Effect of siRNA-mediated inactivation of SFRS2 on splicing. A, Western blot analysis for UMSCC12 and UMSCC22b cells transfected with SFRS2 siRNA or scrambled control sequence. B, bar diagram with percentage of aberrantly spliced transcript in cells transfected with exon 11 minigene construct and SFRS2 siRNA as determined by transcript-specific real-time PCR in UMSCC12 and UMSCC22b. Control cells were transfected with the exon 11 minigene construct and scrambled siRNA. Data are mean ± SD, n = 3. C, E-cadherin expression by real-time PCR analysis in 2 cell lines transfected with varying amounts of SFRS2 siRNA.
- Figure 6.
Hypomethylating agent and aberrant splicing. A, methylation-specific PCR for UMSCC1 and UMSCC22b cell lines with and without azacytidine treatment. B, Western blot analysis for UMSCC1 and UMSCC22b showing E-cadherin expression and actin control with azacytidine treatment. C, expression of wild-type E-cadherin transcripts relative to the untreated cells and adjusted to actin, along with the percentage of aberrant transcript after azacytidine treatment.
Additional Files
Supplementary Data
Files in this Data Supplement:
- Supplementary Table 1 - XLS file - 13K, List of 100 top most downregulated genes in the array. Average of 5 cell H&N cell lines compared to Tert Kert cells
Volume 10, Issue 9
