Figure 6.

The role of the E12A variant in gene regulation and tumor cell survival. A, for global profiling of target genes, RNA-seq experiments were performed on HeLa cells with HNRPLL or E12A knockdown (see “Experimental procedures”). The heat map represents the distribution of genes differentially expressed compared with the control culture. The expression values were displayed in shades of red or blue relative to the means of all corresponding values within individual experimental groups. Hierarchical clustering was performed on the samples by Euclidian distance and average linkage method. B, vectors expressing control (pSUPER) or E12A-specific (shE12A) shRNAs were transfected into HeLa cells, and RNA expression of the indicated genes was analyzed by qPCR assay (n = 5). C, subcellular distribution of the CCND1 and TGFBR1 RNA transcripts in the indicated transfected HeLa cells was examined by qPCR experiment (n = 3). D, nascent RNA synthesis of the indicated genes in HeLa transfectants was determined by qPCR assay (n = 4). E, expression of the CCND1 protein in E12A-knockdown HeLa cells was analyzed by Western blotting assay, with GAPDH as loading control. F, HeLa cells with E12A-knockdown were harvested and subjected to ChIP assay, using control IgG or Pol II antibody. The abundance of the precipitated chromatin complexes was quantified by qPCR with a specific primer to the CCND1 promoter sequence (n = 3). G, colony formation assay was performed on E12A-knockdown HeLa cells, and images of colony formation assay shown in the right panel (representative sample, n = 2). Starting cell number in each experiment was noted. H, vectors expressing control (pSUPER) or E12A-specific (shE12A) shRNAs were transfected into HeLa cells, which were subsequently treated with different doses of doxorubicin, as indicated. The treated cells were collected for PARP protein expression analysis via Western blotting, and the cleaved PARP ratio (CI) was quantitatively determined by GelQuantNet software. GAPDH was used as loading control. I, to assess the apoptotic consequence of E12A mis-expression, HeLa cells were transfected with the control or E12A-targeting shRNA-expressing vector, together with E12A-specific RNA-only (E12A-RNA) and protein (E12A-protein) expression vectors, as indicated. The transfected cells were treated with the indicated dose of doxorubicin, and subsequently collected for PARP protein expression analysis as shown in H. GAPDH expression serves as internal control.