**2.4 Transient transfection assay**

Transient transfection of Luc-reporter plasmids was performed using multi-well culture plates that had been prepared and treated with DNA/DEAE-dextran (Uchiumi et al., 2010a). After 4 h of transfection, 2DG or Rsv was added to the culture medium (Zhou et al., 2009; Uchiumi et al., 2011). After a further incubation (19 to 24 h), cells were collected and lysed with 40 μL of 1 x Cell culture lysis reagent, mixed, and stored at -80°C. Luc assays were performed according to the manufacturer's instructions (Promega). In brief, Luc assay reagent (40 μL) was added to 10 μL of protein sample and mixed briefly. Immediately after mixing, chemiluminescence was measured for 7.5 sec with a Minilumat LB9506 luminometer (Berthold, Bad Wildbad, Germany). Protein assays were performed with the Luc sample (2.5 μL) and Protein Assay Reagent (Bio-Rad Lab., Hercules, CA, USA).

Characterization of 5'-Flanking Regions of Various

Human Telomere Maintenance Factor-Encoding Genes 589

Fig. 1. Promoter regions of the human genes encoding telomere-associated proteins or shelterin protein factors. PCR-amplified 5'-flanking regions of these genes, which were inserted upstream of the *Luciferase* gene of the pGL4-basic vector (pGL4[luc 2.10]), are shown. Transcription start sites (or 5'-end of cDNAs) are designated +1. The TF-SEARCH program (http://www.cbrc.jp/research/db/TFSEARCH.html) was performed and putative

transcription-factor binding-elements (score > 85) are shown schematically.
