Figure 4.

Compound C induces an increase in TERRA levels in HeLa cells. HeLa cells were treated with dimethyl sulfoxide (DMSO) or compound C (5 µM). DNA, RNA, and proteins were extracted after 18 h of treatment. (A) TERRA from subtelomeres Xq, Yq, 15q, and 9p and TERRA from subtelomeres Xp and Yp was quantified by RT-qPCR using TR/TF and Xp-R/Xp-F primer pairs, respectively; levels were normalized to GAPDH mRNA, and all values were compared to DMSO-treated sample. The bars represent the average values from three biological and two technical replicates for each sample. Error bars represent the standard deviations. p values were calculated by paired two-tailed Student’s t-test (n = 3). ** p < 0.01. *** p < 0.001. (B) Western blot analysis of the phosphorylation of AMP-activated kinase (AMPK) and acetyl-CoA carboxylase (ACC). Tubulin was used as a loading control; 15 µg of total protein was loaded in each well. This result is representative of five independent experiments. (C) Analysis of the methylation of the 29 bp repeats using the method described in Figure 1B. Genomic DNA (500 ng) was digested with HpaII (methyl sensitive) or MspI (methyl insensitive). A sample without DNA (blank) was used as negative control. Elongation products (1/20 of sample) were amplified by PCR (35 PCR cycles). PCR products were run on a 1.5% BET-agarose gel. Ladder is the 50 bp DNA ladder (New England BioLabs).