Fig. 2: lncREST is a lncRNA induced on chromatin by p53 following replication stress. | Nature Communications

Fig. 2: lncREST is a lncRNA induced on chromatin by p53 following replication stress.

From: The chromatin-associated lncREST ensures effective replication stress response by promoting the assembly of fork signaling factors

Fig. 2

a Diagram of lncREST locus and RNA-seq tracks (counts per million) for chromatin and total fraction of HCT116 cells treated with 1 mM HU for 8 h. lncREST consists of three exons, is sense intronic to the protein coding gene NDUFAF6, and is located in proximity of the protein coding gene TP53INP1. b qRT-PCR analysis of lncREST in cellular fractions following treatment with HU. (two-tailed unpaired t-test, mean ± SD, n = 3 per condition) Cytoplasm p = 0.2441 (ns); nuclear p = 0.0111 (*); chromatin p < 0.0001 (****). c Representative images of lncREST FISH in HCT116 cells treated with PBS or HU o.n. and dot plot of lncREST foci quantification, mean ± STD. Three experiments were performed with similar results, at least 80 cells per condition were analyzed (n = 2). ****p < 0.0001 (two-tailed unpaired t-test). Scale bar: 10 µm. Uncropped images are provided with the Source Data file. d From top to bottom: Flow cytometry profiles of HCT116 synchronized cells, representing the DNA content at different cell cycle stages from release; relative expression of lncREST and cyclin E measured by RNA-seq. log2FC of each time point vs all other time points is represented. *p < 0.05, **p < 0.01, ***p < 0.001 (p = 0.017243161 for lncREST in early S; p = 0.036741047 for lncREST in late S; for cyclin E p = 0.000486891, p = 0.00130681, p = 0.000137981 in G1, early S and G2, respectively; western blot of cell cycle and DNA damage markers in each time point. Source data are provided as a Source Data file. e qRT-PCR analysis of lncREST expression in HCT116 cells treated with 2 mM of hydroxyurea (HU) o.n., 40 µM aphidicolin (APH) for 24 h, 15 µM of cisplatin (cis) o.n., 5 µM of doxorubicin (Dox) for 24 hrs, 20 µM of nutlin (Nut) o.n., 10 µM of camptotecin (CPT) for 8 h, relative to normal condition (NT). n = 3 biological replicates. **p < 0.01, ***p < 0.001, ****p < 0.0001 (mean ± SD, two-tailed unpaired t-test). f Integrative genomics viewer (IGV) browser snapshot of p53 ChIP-seq enrichment in control and 5-FU treated HCT116 cells. g qRT-PCR of lncREST in HCT116 wt, HCT116 p53−/−, and H358 p53−/− cells untreated, HU and CPT treated. (mean ± SD, two-tailed unpaired t-test. n = 3) (HCT116 p53−/− *p = 0.0324 for HU treatment, H358 **p = 0.0026 for CPT treatment, HCT116 wt **p = 0.0012 for HU treatment, ****p < 0.0001 for CPT treatment. For b–e and g, source data are provided as a Source Data file.

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