Telotaggg telomere length assay

Telomere terminal restriction fragment (TRF) analysis was performed using a commercial kit (TeloTAGGG Telomere Length Assay, 12209136001 ;Roche Life Science). Genomic DNA from the cell lines was isolated with DNeasy Blood & Tissue kit (69504; Qiagen), and 1.5 μg DNA for each sample was digested using HinfI and RsaI restriction enzymes. Digested DNA underwent electrophoresis through a 0.8% agarose gel (111860; Biowest, Nuaille, Maine‐et‐Loire, France) for 4 h at 6 V·cm⁻¹ in 1× Tris/acetate/EDTA (TAE) buffer. Gels were denatured, neutralized and transferred to positively charged nylon membranes (RPN2020B, GE Healthcare) overnight. The membranes were hybridized in DIG Easy Hyb Granules (11796895001; Roche Diagnostics, Penzberg, Germany) containing the telomere probe at 42 °C overnight. The TRF length was quantitatively measured according to the kit instructions.

A 2-mg aliquot of DNA was digested with Fast Digest HinfI (4 μl/2 mg of DNA; Thermo Fisher Scientific) at 37 °C for 2 h. Digested DNA samples were loaded onto 0.8% agarose gels and were run at 80 V for 4 h. To assess telomere length, Southern blotting was performed with a commercial kit (TeloTAGGG Telomere Length Assay; Roche), in which a 5′-TTAGGG-3′ digoxigenin-labelled telomere probe is used and visualised by linking to a chemiluminescent substrate. Blots were imaged in an image analyser (ImageQuant 350; GE Healthcare). We compared two different methods of analysing telomere shortening: the more established method, based on the average telomere lengths (i.e. the mean number of terminal restriction fragments (TRFs)); and a newer method, in which telomeres are stratified by length and the proportions of short (<8.6 kb), medium (8.6–21.2 kb) and long (>21.2 kb) telomeres are determined.

Terminal restriction fragment (TRF) analysis was performed according to the manufacturer's instructions with minor changes (TeloTAGGG Telomere Length Assay – Roche Applied Science, Mannheim, Germany). Briefly, genomic DNA (800 ng) was digested by an optimized mixture of HinfI and RsaI FastDigest restriction enzymes (Thermo Scientific, Waltham, MA, USA) at 37°C for 2 h. Following DNA digestion, DNA fragments were separated by electrophoresis in 0.8% agarose gel during 5 h. Gel was denatured, neutralized, and samples were transferred to a nylon membrane by Southern blotting and probed. Terminal restriction fragments were detected by chemiluminescence. Mean TRF length was determined according to the formula TRF = Σ(ODi)/Σ(ODi/Li), where ODi is the chemiluminescent signal and Li is the length of the fragment at a given position. In every experiment, the mean TRF of a reference sample was determined in order to validate the results.

The average TRF length was determined according to the commercial kit (TeloTAGGG Telomere Length Assay, 12209136001; Roche Life Science). Genomic DNA was extracted by traditional phenol:chloroform:isoamyl alcohol method. 3 μg DNA was digested with MboI (NEB) overnight, and the DNA fragments were separated by 0.8% agarose gel for 16 h at 6 V/cm in 0.5× TBE buffer using CHEF DR-III pulse-field system (Bio-Rad). Gels were denatured, neutralized, and transferred to nylon membranes (RPN2020B; GE Healthcare) for 48 h. The membrane was hybridized with digoxigenin (DIG)-labeled telomere probe at 42 °C overnight and incubated with the anti-DIG-alkaline phosphatase antibody. Telomere signals were detected by chemiluminescence after the addition of the substrate solution on membrane. Telomere length was measured by TeloTool software.

2.2 × 10⁶ cells were plated on 10 cm plates for each cell line with DMEM (10% FBS). Cells were plated with or without doxycycline depending on the condition. Cells were split 1:3 on Day 3 and Day 5. Adherent cells were harvested on Day 1 Dox and Day 8 Dox/No Dox, taking care of eliminating floating cells. DNA from cell pellets was extracted using the PureLink™ Genomic DNA Mini Kit (Invitrogen) according to the manufacturer’s instructions. Mean telomere lengths of samples were assessed using the TeloTAGGG™ Telomere Length Assay (Roche) according to manufacturer’s instructions aside from modifications listed. For each sample, 4 μg of DNA was digested with Hinf I/Rsa I enzyme mixture. An overnight (14 h) capillary transfer setup was used to transfer the DNA to BrightStar™—Plus positively charged nylon membrane (Invitrogen) using 20X SSC transfer buffer. A DNA crosslinker was used to fix the DNA on the nylon membrane following overnight transfer. Chemiluminescent images were taken using a ChemiDoc™ MP Imaging System. ImageLab was used to assess average telomere lengths for each sample. Graphs were created using GraphPad Prism9. An ordinary one-way ANOVA with multiple comparisons was used to assess statistical significance of average telomere length changes.

TRF analysis was performed using a commercial kit (TeloTAGGG Telomere Length Assay, catalog no. 12209136001, Roche Life Science). Cells were pretreated with RNaseA and Proteinase K (PCR Grade, 03115879001, Roche Life Science), followed by extraction using phenol: chloroform: isoamyl alcohol, digested with MboI (R0147, NEB) at 37 °C overnight and electrophoresed through 1% agarose gels in 0.5 × TBE at 14 °C using a CHEF Mapper pulsed field electrophoresis system (Bio-rad). Auto algorithm was used to separate DNA samples with a size range from 5 to 150 kb. The gel was blotted and probed using reagents in the kit.