Fig. 3: lncREST regulates replication stress response. | Nature Communications

Fig. 3: lncREST regulates replication stress response.

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

Fig. 3

a qRT-PCR of lncREST in HCT116 cells following depletion with two independent LNA GapmeRs. ****p < 0.0001 (mean ± SD, two-tailed unpaired t-test. n = 3). b Percentage of cells in each cell cycle fraction measured by flow cytometry in cells treated with 1 mM HU o.n and 40μM aphidicolin (APH) 24 h. *p < 0.05, **p < 0.01 (mean ± SD, two-tailed unpaired t-test. n = 3). c Alkaline comet assay showing the increase ss-and dsDNA breaks in lncREST KD cells in NT condition and challenged with 1 mM HU o.n. and 40μM aphidicolin (APH) 24 h. Left panel, representative images. Scale bar, 100 µm. Right panel, boxplots showing the alkaline olive moment. Whiskers are set to min/max value, boxes extend from the 25th to 75th percentiles, median is shown. At least 200 tails were analyzed for each condition (n = 3). **p = 0.0081 for CTRL KD vs lncREST KD1 NT, ***p = 0.0004 for CTRL vs lncREST KD1 HU, ****p < 0.0001 (two-tailed unpaired t-test). d Micronuclei in lncREST KD cells. Left panel, representative images. Right panel, percentage of cells with one or more micronuclei from three independent experiments. *p = 0.011553 for lncREST KD1 vs CTRL HU, *p = 0.017958 for lncREST KD2 vs CTRL NT, *p = 0.016264 for lncREST KD2 vs CTRL HU, (mean ± SD, two-tailed unpaired t-test. n = 3 per group). e Western blot of indicated proteins in CTRL and lncREST KD cells with two LNA GapmeRs treated with 1 mM HU o.n. Numbers indicate relative protein quantity normalized to GAPDH. Experiments were performed at least two times for each antibody indicated with similar results. Source data are provided as a Source Data file. f, g Representative images and quantification (dotplot and median) of γH2A.X f and p-ATR g foci per nucleus in HCT116 cells transfected with CTRL or lncREST LNAs in NT cells and after treatment with HU 1 mM 3 h and o.n. Scale bar, 10 μm. At least one hundred cells were analyzed per sample in two or three independent replicates. ***p = 0.0004 for lncREST KD2 vs CTRL NT γH2A.X, ****p < 0.0001 (mean ± SD, two-tailed Mann Whitney U-test). For a, c–f, and h, source data are provided as a Source Data file.

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