Fig. 7: lncREST regulates proliferative phenotypes and is required for accurate mitosis. | Nature Communications

Fig. 7: lncREST regulates proliferative phenotypes and is required for accurate mitosis.

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

Fig. 7

a Score of the replication stress CX9 signature40 in TCGA colorectal tumors with high and low lncREST expression. TCGA patient samples were divided in two groups depending on their lncREST expression (40% upper percentile as high, 60% lower percentile as low). For each one of them, the activity of the 17 copy number signatures was calculated and plotted. Boxplots represent 25 to 75 percentiles, whiskers are 1.5 x interquartile range (interquartile range = percentile75-percentile25). b Mitotic HCT116 cells stained with α-tubulin (green) and p-CENP-A (red) antibodies and transfected with CTRL or lncREST targeting LNA GapmeRs, and relative quantification showing the percentage of mitotic cells identified for each type of alignment. Representative image of the predominant type of alignment for CTRL and lncREST KD cells, are shown. 50 cells per sample were analyzed. n = 3 for CTRL and lncREST KD1, 2 for lncREST KD2. *p < 0.05; **p < 0.01; ***p < 0.001. (mean with SD, two-tailed unpaired t-test). c, d Cell proliferation measured my MTS assay in HCT116 cells (untreated and HU-treated) transfected with lncREST of CTRL LNA GapmeRs c and lncREST KO (two-tailed unpaired t-test, mean ± SD, n = 3). *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001 d. Absorbance (Abs) at 490 nm was measured over a time course of 4 days, starting from 48 h post transfection in (c), and the day after plating the cells in (d). *p < 0.05; **p < 0.01; ***p < 0.001; (mean ± SD, n = 3 per group). e, f Analysis of tumors generated by subcutaneous injection of control or lncREST-depleted HCT116 cells e or lncREST KO cells f in BALB/cA-Rag2 − /−γc − /− mice. Graphs show average tumor volume ±STD calculated using the formula Length x Width x Height/2. two-tailed unpaired t-test (n = 4 for e, *p = 0.0012 T1, *p = 0.035 T3, *p = 0.027 T4, **p = 0.017 T6, *p = 0.049 T7. n = 6 for f, *p = 0.036 T2, **p = 0.0028 T3, ***p = 0.00015 T4. g Proposed model for the mechanism of action of lncREST. Upon replication stress, NCL protein localizes to the chromatin where it interacts with lncREST. This is required for the localization of RPA and p-ATR at the forks for the effective S-checkpoint signaling, leading to fork stalling. The action of lncREST protects cells from genomic instability by assuring the correct functioning of the S-phase checkpoint. For b–f, source data are provided as a Source Data file.

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