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. 2014 Jan 8;9(1):e84931. doi: 10.1371/journal.pone.0084931

Figure 1. Depletion of SIRT1 did not affect TERT promoter activity.

Figure 1

(A) A schematic representation of TERT promoter reporter plasmids. The TERT promoter reporter plasmids were generated by inserting the 1.0 kb and 0.4 kb DNA fragments of TERT promoter upstream of the initiating ATG or 0.4 kb core promoter as well as the first exon of TERT into luciferase (Luc) reporter vector pGL3-Basic in the sense orientation. Arrow, the transcription start site. Numbers, the number of bases upstream (-) and downstream (+) of the translational start codon. (B) Western blot shows the TERT reduction following SIRT1 suppression in SK-HEP-1 and PLC5 cell lines. (C) and (D) Sub-confluent cultures of SK-HEP-1 or PLC5 cells expressing the indicated shRNA in 24-well plate were transfected with indicated luciferase reporter plasmids along with a pRL-TK reporter plasmid as a control for transfection efficiency. The pGL3-SV40 containing the minimal, enhancerless SV40 promoter was used as a control. After 48 h of incubation, cells were lysed and the lysates were analyzed for dual luciferase activity. Normalized relative luciferase activity in shCont-expressing cells was designated as 1.0. Results are the mean+/−S.D. of triplicate measurements from one of three representative experiments with similar results. Statistical analysis was performed with Student's t-test.