Figure 7: Southern blot analysis of WT and mutant POT1.

(a) Quantitative RT-PCR showing knock down of endogenous POT1 mRNA, normalized to GAPDH transcript levels, after lentiviral infection of HEK293T cells with shPOT1 and WT Flag-POT1; selection was carried out with puromycin and blasticidin;±s.d. (n=3). (b) Western blot showing WT and mutant POT1 protein expression levels in HEK293T cells expressing shPOT1 and Flag-POT1. Cells infected with the empty pLU and pLKO.1 lentiviral vectors are used as controls. (c,d,e) Southern blot of HEK293T cells expressing shPOT1 and WT or mutant flPOT1. DNA from 5, 50 and 120 populations doublings (PD) is shown. DNA length standards are indicated along the left and right of the gels. The white dashed line indicates the average telomere length of HEK293T cells transfected with the vector alone and no shPOT1 after 5, 50 and 120 PD. The vector (- shPOT1), vector (+shPOT1) and WT POT1 (+shPOT1) controls are indicated with a green and yellow dot respectively. The POT1 mutants (L343F, P466Q, P475L, R477T, A532P, I535F, C591W and Q623H) are indicated with red dots. (f) Quantification of the mean telomere length (kb) from panels (c,d and e). The same colour scheme as in panels c,d and e is used. Black bars indicate the range of telomere restriction fragments (TRF).