u2os dr gfp cell line  (ATCC)

Journal: bioRxiv

Article Title: Cell-cycle dependent inhibition of BRCA1 signaling by the lysine methyltransferase SET8

doi: 10.1101/2023.11.22.567520

Figure Lengend Snippet: ( A ) Representative images of RAD51, BRCA1 and 53BP1 staining in EdU positive nuclei from control (-Tet) and FLAG-Set8 PIPmut (+Tet) expressing cells treated with 5 or 25 nM of camptothecin during 2 hours or with vehicle (DMSO) as indicated. Scale bar = 10 µm. ( B ) Scattered box-plot representing the quantification of RAD51, BRCA1 and 53BP1 foci per EdU-positive nuclei from control (-Tet) and FLAG-Set8 PIPmut expressing cells (+ Tet) treated with 5 nM or 25 nM of camptothecin during 2 hours, or with vehicle (DMSO). Interquartile range and statistical significance are shown as in . n ≥ 3; *** p<0.001 (t test). Number of nuclei per condition and experiment >100. ( C ) Left panel: Bar-plot representing the relative efficiency of DNA repair by homologous recombination of I-SceI-induced DNA breaks as evaluated by flow cytometry using the U2OS DR-GFP cell line after expression of different FLAG-SET8 proteins as indicated. Data = mean ± s.d., n = 3. *** p<0.001 (t-test). Number of events per sample > 10 000. Right panel: immunoblots showing the similar expression of different FLAG-SET8 proteins and of MYC-tagged nuclease I-SceI in U2OS DR-GFP cells. Tubulin was used as loading control.

Article Snippet: The HR assays were performed using the U2OS DR-GFP cell line (ATCC) that allows to monitor homologous recombination efficiency through the detection of GFP positive cells via flow cytometry.

Techniques: Staining, Control, Expressing, Homologous Recombination, Flow Cytometry, Western Blot

Journal: bioRxiv

Article Title: Cell-cycle dependent inhibition of BRCA1 signaling by the lysine methyltransferase SET8

doi: 10.1101/2023.11.22.567520

Figure Lengend Snippet: ( A ). Representative immunoblots showing the localization of BRCA1 and 53BP1 in soluble cytoplasmic (S1) and nuclei (S2) fractions and in chromatin-enriched fractions (P3) at early G1 phase in control and SET8 SiRNA-treated cells that excited from mitosis without SET8 and the conversion of H4K20me0 to H4K20me1 as shown in supplementary figure S7. Cells were harvested 15 hours after G1/S release. n=3 ( B ) FACS analysis of DNA content and BrdU signal after mitotic exit of the control and SET8 SiRNA-treated cells as described above. Cells were harvested 24 hours after G1/S release. n=3 ( C ) Representative images showing EdU incorporation (DNA synthesis) and staining of 53BP1 and BRCA1 in control and siRNA SET8 treated cells progressing from G1 to S-phase after a first mitosis without SET8 and H4K20me1 as described above. Scale bar = 10 µm ( D ). FACS analysis of DNA content and BrdU incorporation levels of control (shLuc/siCtrl), SET8-depleted U2OS cells (shRNA SET8/siRNA Ctrl), BRCA1-depleted U2OS cells (shRNA Luc/siRNA BRCA1) and cells depleted for both BRCA1 and SET8 (shRNA SET8/siRNA BRCA1). n=3. Number of of events per sample > 10 000.

Article Snippet: The HR assays were performed using the U2OS DR-GFP cell line (ATCC) that allows to monitor homologous recombination efficiency through the detection of GFP positive cells via flow cytometry.

Techniques: Western Blot, Control, DNA Synthesis, Staining, BrdU Incorporation Assay, shRNA