Fig. 2: p53-induced LOC644656 is required for genotoxic stress-mediated suppression of pluripotency in hESCs.

a Venn diagram of 268 differentially expressed (DE) lncRNAs identified from RNA-seq and p53 ChIP-seq analyses (DDR vs DMSO > 5-fold). b Integrative Genomics Viewer tracks for p53 ChIP-seq, ATAC-seq, and RNA-seq at the LOC644656 locus. The pink asterisk indicates the α-p53 ChIP-seq track in p53KO hESCs. c, d Immunoblot analyses of p53 after treatment with Nutlin-3a (c) or 5-FU (d). Molecular weight markers (kDa) are shown on the left. Blots are representative of n = 3 independent experiments with similar results; uncropped blots are provided in the Source Data file. The samples derive from the same experiment but different gels for p53 and β-actin in parallel. e–h Real-time RT-PCR analysis of p21/CDKN1A (e, g) and LOC644656 (f, h) relative to ACTB under Nutlin-3a or 5-FU treatment. Data are presented as mean ± SEM from n = 3 biologically independent samples. *p < 0.05, **p < 0.01, ***p < 0.001 (two-sided one-way ANOVA with Dunnett’s post hoc test). i, RNA-FISH of LOC644656 in hESCs ± 5-FU. Yellow arrows indicate nuclear speckles. Images are representative of n = 3 independent experiments with similar results. Scale bars: 100 μm (main images), 20 μm (insets). j Subcellular fractionation of LOC644656 in cytoplasmic (Cyt), nuclear (Nuc), and chromatin (Chr) fractions. XIST is used as a nuclear control. *Data are mean ± SEM (n = 3), p < 0.05 by two-sided Student’s t-test vs untreated. k Schematic of CRISPR/Cas9-based LOC644656 knockout (KO). l LOC644656 expression in WT vs KO hESCs. **Data are mean ± SEM (n = 4), **p < 0.0001 by two-sided Student’s t-test. m NANOG expression ± 5-FU in WT vs KO hESCs. **Data are mean ± SEM (n = 3); two-way ANOVA (two-sided) with Tukey’s post hoc test, **p < 0.01, *p < 0.001 vs untreated WT. n FITC-rBC2LCN staining of pluripotency ± 5-FU for 12 h. Images are representative of n = 3 independent experiments with similar results. Scale bar, 100 μm. Exact p-values and 95% confidence intervals: Fig. 2e: p = 0.017, 95% CI [16.10, 114.3] (0 μM vs 30 μM p53WT); p < 0.0001, 95% CI [86.74, 185.0] (0 μM vs 50 μM p53WT); Fig. 2f: p < 0.0001, 95% CI [−6.118, −2.894] (0 μM vs 30 μM p53WT); p < 0.0001, 95% CI [−12.29, −9.061] (0 μM vs 50 μM p53WT); Fig. 2g: p = 0.0391, 95% CI [−71.64, −4.455] (0 μM vs 300 μM p53WT); p = 0.0055, 95% CI [−82.77, −12.59] (0 μM vs 1000 μM p53WT); Fig. 2h: p < 0.0001, 95% CI [−3.369, −1.235] (0 μM vs 100 μM p53WT); p < 0.0001, 95% CI [−3.852, −1.719] (0 μM vs 300 μM p53WT); p < 0.0001, 95% CI [−4.994, −2.860] (0 μM vs 1000 μM p53WT); Fig. 2j: p = 0.0341, 95% CI [2.451, 24.09]; Fig. 2l: p = 0.001, 95% CI [−1.091, −0.8315]; Fig. 2m: p = 0.0052, 95% CI [−0.8581, −0.1572] (LOC644656 WT vs KO, 8 h); p = 0.001, 95% CI [−0.9778, −0.2769] (LOC644656 WT vs KO, 24 h).