Fig. 6: lncREST interacts with NCL and modulates RPA association to chromatin. | Nature Communications

Fig. 6: lncREST interacts with NCL and modulates RPA association to chromatin.

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

Fig. 6

a RNA immunoprecipitation with NCL antibody shows lncREST-NCL association. The upper panel shows successful NCL pulldown by western blot in NT and HU-treated cellular lysates, and relative inputs. The lower panel shows qRT-PCR detection of lncREST precipitated by NCL. **p = 0.0093 ACTB HU, **p = 0.0047 CONCR HU, ****p < 0.0001 lncREST HU (mean ± SD, two-tailed unpaired t-test. n = 3). b In vitro RNA pulldown assay revealed a preferential binding of NCL to the lncREST fragment containing the 3’ end. Antisense lncREST sequence was used as control. Results were confirmed by three independent experiments. c Upper panel, Western blot of NCL in cellular fractions of HCT116 NT and treated with 1 mM HU o.n. Bottom panel, quantification of NCL on each fraction relative to H3 or GAPDH. **p = 0.007081 (mean ± SD, two-tailed unpaired t-test. n = 3). d Left panel, Western blot of chromatin fractions of lncREST depleted HCT116 cells shows reduction of chromatin-associated NCL and RPA32 in 1 mM HU treated cells. Right panel, quantification of NCL and RPA32 on chromatin fraction relative to H3. For NCL **p = 0.001745, ****p < 0.0001; for RPA32 *p = 0.030133, ****p < 0.0001 (mean ± SD, two-tailed unpaired t-test. n = 3). Source data are provided as a Source Data file. e IF of NCL in lncREST depleted and control HCT116 cells in NT and HU treated condition (2 mM for 5 h) showing a reduction of the nuclear pool of NCL in KD cells. ***p = 0.0005, ****p < 0.0001 (mean ± SD, two-tailed unpaired t-test. n = 3). f Immunofluorescence of chromatin-associated RPA32 of HCT116 transfected with CTRL and lncREST LNA GapmeRs, following HU treatment as indicated, after removal of soluble cellular fraction before fixing the cells, and dot blot (two-tailed unpaired t-test, mean ± SD) of RPA32 fluorescence normalized by nuclear area. At least 70 cells for each biological replicate were analyzed. **p = 0.0086, ****p < 0.0001 (two-tailed unpaired t-test. n = 3 per group). g Western blot (left) and quantification (right) of CTRL LNA or lncREST KD cells- NCL co-immunoprecipitated with RPA32 or a control IgG in untreated or HU-treated cells. **p = 0.0043 in NT, **p = 0.0098 in HU (mean ± SD, two-tailed unpaired t-test. n = 4). Source data are provided as a Source Data file.

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