Journal: Nucleic Acids Research
Article Title: RSRC2 is a novel RNA-binding protein that safeguards mitotic fidelity by interacting with the lncRNA C1QTNF1-AS1
doi: 10.1093/nar/gkag229
Figure Lengend Snippet: Loss of C1QTNF1–AS1 or RSRC2 results in increased mitotic index and intact SAC signalling. ( A ) Representative images of metaphase cells stained for kinetochores (CREST, cyan), SAC (Mad2, green), and DNA (Hoescht, magenta) after LNA-mediated depletion of C1QTNF1–AS1 in HCT116 cells. ( B ) Insets show examples of MAD2 and CREST staining from C1QTNF1–AS1 -depleted cells, as in panel (A); scale bar: 0.5 µm. ( C ) Characterization of SAC activity in HCT116 cells after LNA-mediated depletion of C1QTNF1–AS1 . Percentage of mitotic cells with Mad2 signal on misaligned chromosomes using the same antibodies as in panel (A); N = 3 ( n (Ctl A) =67; n ( C1QTNF1–AS1 LNA1) = 69; n ( C1QTNF1–AS1 LNA2) = 72; n ( C1QTNF1–AS1 LNA3) =93). ( D ) Representative images of metaphase HCT116 cells stained for kinetochores (CREST, cyan), SAC (Mad2, green), and DNA (Hoescht, magenta) following siRNA-mediated depletion of RSRC2. ( E ) Insets show examples of MAD2 and CREST staining from RSRC2-depleted cells, as in panel (D); scale bar: 0.5 µm. ( F ) Characterization of SAC activity in HCT116 cells after siRNA-mediated depletion of RSRC2. Percentage of mitotic cells with Mad2 signal on misaligned chromosomes using the same antibodies as in panel (C); N = 4 ( n (Ctl si) = 108; n (RSRC2 si) = 126). ( G ) Quantification of mitotic duration from time-lapse imaging microscopy following LNA-mediated depletion of C1QTNF1–AS1 (left panel) and siRNA-mediated depletion of RSRC2 (right panel) in HCT116 H2B–GFP cells treated with nocodazole. N = 3 ( n (Ctl A) = 49; n ( C1QTNF1–AS1 LNA1) = 47; n ( C1QTNF1–AS1 LNA2) = 50; n ( C1QTNF1–AS1 LNA3) =51, n (Ctl si) = 87; n (RSRC2 si) = 51). ( H ) Changes in mitotic index after LNA-mediated depletion of C1QTNF1–AS1 (left panel) and siRNA-mediated depletion of RSRC2 (right panel) in HCT116 cells. Percentage of cells in mitosis shown based on Histone–H3–pS10 staining; N = 3 ( n (Ctl A) = 119; n ( C1QTNF1–AS1 LNA1) = 141; n ( C1QTNF1–AS1 LNA3) = 136; n (Ctl si) = 129; n (RSRC2 si) = 204). Error bars in all panels are shown as mean ± S.E.M.; scale bar: 5 μm. An unpaired t -test with Welch’s correction was applied in panels (C) and (F). The Mann–Whitney test was used in panel (G). A one-way ANOVA (left panel) and an unpaired t -test (right panel) were used in panel (H).* <0.05, **<0.01, ***<0.001 and ****<0.0001.
Article Snippet: Human normal retinal pigment epithelial hTERT-RPE1 (ATCC) and hTERT-RPE1 H2B-GFP (provided by Prof. David Pellman, USA) cells were maintained in Dulbecco's modified Eagle's medium (DMEM) F12 medium (Sigma, D8437), supplemented with 10% foetal bovine serum (FBS, A5256801, Gibco).
Techniques: Staining, Activity Assay, Imaging, Microscopy, MANN-WHITNEY
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