Journal: Nucleic Acids Research

Article Title: MDS/AML-associated DDX41 helicase facilitates homologous recombination repair by potentially resolving R-loops

doi: 10.1093/nar/gkag219

Figure Lengend Snippet: DDX41 loss diminishes HR repair. ( A ) Western blot assays of DDX41 siRNA and siRNA control in U2OS GFP reporter cell lines with indicated antibodies. β-Actin serves as a loading control. Quantification of relative protein level for DDX41 and I-SecI is shown on the right. ( B ) Percentages of GFP positive cells as assessed by flow cytometry 36 h after U2OS GFP reporter cell lines treated with siRNA control, siRNA control + I-SceI plasmid, or DDX41 siRNA + I-SceI plasmid. ( C ) Cell survival assays of WT and DDX41–KO HT1080 cells treated with Olaparib. ( D and E ) Immunofluorescence staining of WT and DDX41–KO HT1080 cells with γH2AX, RPA32 (D) or RAD51 (E), and DAPI without bleomycin treatment (UT) or 4 h post BLM treatment (30 μg/ml for 1 h). Quantification of RPA32 and RAD51 foci is shown in the middle. Data represent the mean ± SEM of three independent experiments. * P < 0.05, *** P < 0.001, and **** P < 0.0001.

Article Snippet: U2OS (HTB-96, ATCC) and U2OS GFP reporter cells [ ] (a gift from Jeremy Stark, City of Hope) were grown in Mccoy 5A media (16600082, Thermo Fisher) supplemented with 10% fetal bovine serum.

Techniques: Western Blot, Control, Flow Cytometry, Plasmid Preparation, Immunofluorescence, Staining

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