Figure 7.

HOT1 acts as a positive regulator of telomere length. (A) Verification of HOT1 esiRNA knockdown efficiency using a HOT1-LAP cell line (Poser et al, 2008) as a reporter for HOT1 expression by western blot after 2, 3, 4 and 6 days. Cells analysed after 4 and 6 days were transfected twice: After the initial transfection, cells were transfected a second time on day 3 (72 h post transfection). All samples were run on the same gel, irrelevant lanes were spliced out. (B) Quantification of telomere length by quantitative telomeric FISH after transient knockdown of HOT1. The distributions of fluorescence intensities, in arbitrary units of fluorescence (a.u.f.), of individual telomeres from a total of 15–20 metaphases per treatment are displayed; the average intensity is indicated in red. Changes of average telomere signal intensity are shown relative to the RLuc (Renilla Luciferase) control. (C) Verification of FLAG–HOT1 and FLAG–HOT1ΔHomeobox expression by western blot 48 h post transfection. (D) Results of telomere length measurements after transient overexpression of FLAG–HOT1 and FLAG–HOT1ΔHomeobox. The distributions of fluorescence intensities, in a.u.f., of individual telomeres from a total of 30 metaphases per treatment are displayed; the average intensity is indicated in red. (E) Sequence-specific pull-down of FLAG–HOT1 and FLAG–HOT1ΔHomeobox. Proteins were incubated with either telomeric repeats (5′-TTAGGG-3′) or a control oligonucleotide (5′-GTGAGT-3′).

Source data for this figure is available on the online supplementary information page.

Source data for Figure 7a ^{(836.7KB, pdf)}

Source data for Figure 7c ^{(1,022.6KB, pdf)}

Source data for Figure 7e ^{(1.6MB, pdf)}