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. 2014 Mar 25;24(5):513–531. doi: 10.1038/cr.2014.35

Figure 3.

Figure 3

In cis overexpression of CCAT1-L enhances MYC expression and tumorigenesis. (A) A schematic view of the strategy to in cis express CCAT1-L in HCT116 cells by TALEN. TALEN A, the CCAT1-L in cis overexpression cell line. A cassette of CMV promoter and sequences of puromycin and egfp mRNAs was inserted just upstream of the first exon of CCAT1 by TALEN. TALEN B, the control cell line that overexpresses egfp. The same cassette of TALEN A but with two additional poly(A) sites to terminate the transcription downstream of egfp was inserted into the same genomic location as that in TALEN A (see Supplementary information, Figure S3 for details). Note that the transcription occurred in both egfp-CCAT1-L- and egfp-overexpressing cell lines. (B) Northern blot validated the overexpression of egfp-CCAT1-L (left panel) or egfp (right panel) in different TALEN lines by using a probe recognizing egfp shown in A. (C) In cis overexpressed egfp-CCAT1-L (B) was poorly translated into EGFP due to its nuclear retention, while the overexpressed egfp was efficiently translated into EGFP. Fluorescence microscopy (top) and WB (bottom) of representative TALEN A or TALEN B lines are shown. (D) Nuclear-retained egfp-CCAT1-L exclusively accumulated as a single nuclear dot, as revealed by ISH probes recognizing either CCAT1-L or egfp in the representative TALEN A clone. (E) Northern blot validated that egfp-CCAT1-L can be efficiently knocked down by the ASO that targets CCAT1-L, assayed with a probe recognizing egfp. (F) RT-qPCR revealed the overexpression of CCAT1-L in TALEN A lines, but not in control TALEN B lines (normalized to actin and the non-engineered HCT116 cells). Four lines of TALEN A and TALEN B cells were analyzed individually. (G) In cis overexpression of egfp-CCAT1-L enhanced MYC expression, as revealed by RT-qPCR (normalized to actin and the non-engineered HCT116 cells). The same lines of TALEN A and TALEN B cells in F were analyzed. (H) In cis overexpression of egfp-CCAT1-L increased tumor formation in a mouse xenograft model. Xenograft tumors were collected 4 weeks after inoculation of cells. Left, representative xenograft tumors generated from a TALEN A and a TALEN B line are shown. Right, comparison was made between the egfp-CCAT1-L in cis overexpressing TALEN A lines and the egfp in cis overexpressed TALEN B lines. Note that xenograft tumors raised from individual in cis egfp-CCAT1-L-overexpressing HCT116 cell lines were larger than those raised from control TALEN-engineered cell lines. Error bars in F-H represent ± SD in indicated multiple experiments. P values from one-tailed t-test in the pairwise comparison are shown. 18S and 28S rRNAs were used as loading controls in all northern blots. Supportive data are included in Supplementary information, Figures S1-S4.