Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2024 Jun 17;15:5151. doi: 10.1038/s41467-024-49567-5

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

PMC Copyright notice

Fig. 3 — a, b Etoposide, not ICRF, dramatically increases the levels of total and nascent α-satRNAs. Total and EU-labelled RNAs from HeLa cells treated with DMSO, etoposide (30 μM), or ICRF (20 or 40 μM), were subjected to real-time PCR analysis. Biological replicates (n = 4 for nascent in (a) and n = 3 for the others). c DSB analysis by comet assay. HeLa cells treated with DMSO, Etoposide (Etop, 30 μM), Phleomycin (Phleo, 80 μg/ml), ICRF (20 μM), MMC (5 μg/ml), Cisplatin (Cispl, 20 μg/ml), or CPT (2.5 μM) for 12 h, were analyzed with comet assay. Similar results were observed in two biological replicates. d DSB-inducing agents increase α-satellite transcription. RNAs from HeLa cells treated with DMSO, Phleo (80 μg/ml, 24 h), MMC (indicated, 12 h), or Cispl (indicated, 12 h) were analyzed with real-time PCR. Results for other primers are recorded in Figs. S6d-f. Biological replates (n = 5 for MMC and n = 3 for others). e Etoposide increases α-satellite transcription in a time-dependent manner. RNAs from HeLa cells treated with DMSO or etoposide (2.5 μM) as indicated were analyzed with real-time PCR. Results for other primers are recorded in Fig. S6g. n = 3 biological replicates. f CRISPR-Cas9 generates DSBs specifically on the centromere. Sat-gRNA plasmids were transfected into HeLa cells for 24 h and immunostaining was performed. ~16% of cells had centromeric γH2AX foci. g DSBs generated by CRISPR-Cas9 increase α-satellite transcription. RNAs in (e) were subjected to real-time PCR analysis. n = 4 biological replicates. h ATM inhibition decrease etoposide- induced γH2AX levels. HeLa cells were treated with DMSO, etoposide (30 μM), etoposide plus KU-55933 (10 μM), or etoposide plus caffeine (2 μM) for 12 h and then stained with the indicated antibodies. Similar results were observed in two biological repeats. Quantification was based on one single experiment. Each dot represents one cell. DMSO n = 32, Etop n = 18, Etop+Ku n = 21, Etop+Caff n = 27. i ATM inhibition does not affect etoposide-induced α-satellite transcription. RNAs in (h) were analyzed with real-time PCR. n = 3 biological replicates. All data here are presented as mean values +/− SEM expect for (h, +/− SD). Two-sided Student’s T-test (a, b, d, e, g) and ANOVA followed by pairwise comparisons using Tukey’s test for (h, i). ns, not significant (P > 0.1). Numeric values for P < 0.1 are shown. Source data are provided as Source Data file.