Figure 5

The fusion proteins inhibit telomerase activity and shorten telomeres of Raji cells. (a) PinX1/C–G₄S–9R–G₄S–mBAFF and PinX1/C–9R–mBAFF inhibit telomerase activity. Telomerase-containing extracts from Raji cells were incubated with various concentrations of fusion proteins for 10 min at 4 °C and then subjected to telomerase extension. Telomerase products were separated on 10% polyacrylamide gels, after which the gels were stained with chemiluminescence reagent. Telomerase activity was semiquantified by normalizing the band intensities of the characteristic 6-bp telomerase-specific ladder to that of the 216-bp internal control (IC) using NIH image software. Arrows indicate 216-bp IC. −, telomerase-containing extracts without treatment with any protein; Rnase, telomerase-containing extracts pretreated with RNase. (b) PinX1/C–G₄S–9R–G₄S–mBAFF and PinX1/C–9R–mBAFF shorten telomere length. Raji cells were continuously maintained in culture by splitting the cells and seeding them at 2 × 10⁵ per well in the presence of 100-nM fusion proteins for 30 population doublings. Genomic DNA was isolated and digested with HinfI and RsaI, and Southern blot analysis was performed using a TTAGGG repeat as a probe. The average TRF length of Raji cells was quantified using ImageQuant. −, Raji cell without treatment with protein.