Figures and data
(A) Localisation of BRCA1 and RAD51 after 1h or 2h of I-Ppo1 WT treatment or 2h of I-Ppo1 H98A treatment, respectively, in U2OS cells. (B) Localisation of BRCA1 and RAD51 in siLuc, siTreacle, siTopBP1, and siNBS1 transfected U2OS cells after 2h of I-Ppo1 WT treatment. All scale bars = 10 µm. (C) Quantification of the experiment in (B) showing the number of BRCA1 and RAD51, respectively, nucleolar caps (n= 100 cells). Graphs show a single representative replicate (out of three), and bars represent mean. (D) Co-immunoprecipitation from 293T cells transfected or non-transfected with HA-tagged Treacle and treated with WT I-Ppo1 for 2h. (E) SIM of Treacle and BRCA1 after 2h of I-Ppo1 WT transfection of Treacle-GFP expressing U2OS cells. Scale bar= 5 µm.
(A) SIM of H2AX and BRCA1 in U2OS cells after 2h of I-Ppo1 WT transfection. Scale bar = 5 μm. (B) SIM of U2OS cells stably expressing Treacle-GFP and treated with I-Ppo1 WT for 2h, stained with BRCA1 and MDC1 antibodies. A reconstruction of nucleolar caps using 3D surface is shown. Scale bar = 1μm. (C) Localisation of BRCA1 and RAD51 in WT, ΔMDC1, and H2AXS139A RPE1 cells 2h after I-Ppo1 WT transfection. All scale bars = 10 µm. (D) Quantification of the experiment in (C) showing the number of BRCA1 (upper panel) and RAD51 (lower panel) caps per cell (n= 90 cells). Graph shows a single replicate (experiment was performed in triplicates) and bars represent mean.
(A) A scheme of various MDC1 mutations introduced to ΔMDC1 U2OS cell line. (B) Localisation of BRCA1, RAD51, and mNeon/GFP-MDC1 after 2h of I-Ppo1 WT transfection. U2OS ΔMDC1 cells expressing wild type MDC1 (WT-mNeon), S168A/S196A MDC1 (DM-GFP), SDT → ADA MDC1 (12A-GFP), MDC1 lacking the entire PST repeat region (ΔPST-mNeon), and TQXF → AQXF (AQXF-GFP). All scale bars = 10 μm. (C) Quantification of the experiment in (B) showing the number of BRCA1 (left panel) and RAD51 (right panel) nucleolar caps per cell (n= 100 cells). Graph represents a single replicate (experiment was performed in duplicates for RAD51 and in quadruplicate for BRCA1) and bars represent mean.
(A) Localisation of BRCA1 and RAD51 in doxycycline inducible RNF8 shRNA U2OS cell line treated with or without doxycycline after 2h of I-Ppo1 WT transfection. All scale bars= 10 μm. (B) Quantification of the experiment in (A) showing the number of BRCA1 (left panel) and RAD51 (right panel) nucleolar caps per cell (n= 100 cells). Graph represents a single replicate (experiment was performed in triplicates) and bars represent mean. (C) Localisation of BRCA1 and RAD51 in doxycycline inducible RNF168 shRNA U2OS cell line treated with or without doxycycline after 2h of I-Ppo1 WT transfection. All scale bars= 10 μm. (D) Quantification of the experiment in (C) showing the number of BRCA1 (left panel) and RAD51 (red panel) nucleolar caps per cell (n= 150 cells). Graph represents a single replicate (experiment was performed in triplicates) and bars represent mean.
(A) Localisation of MDC1 and γH2AX in siLuc, siTreacle, siTopBP1, and siNBS1 transfected U2OS cells after 2h of I-Ppo1 WT treatment. All scale bars = 10 µm. (B) Quantification of the experiment in (A) showing the number of MDC1 (left panel) and H2AX (right panel) nucleolar caps per cell (n = 120 cells). Graphs represent a single replicate (experiment was performed in duplicates) and bars represent mean.
(A) Localisation of BRCA1 and RAD51 in parental and DLD1 ΔBRCA2 cells after 2h of I-Ppo1 WT treatment. All scale bars = 10 µm. (B) Quantification of the experiment in (A) showing the number of BRCA1 (upper panel) and RAD51 (lower panel) nucleolar caps per cell (n= 100 cells). Graph represents a single replicate (experiment was performed in duplicates) and bars represent mean. (C) Localisation of BRCA1 and RAD51 in RPE1 ΔTP53 and RPE1 ΔTP53 ΔBRCA1 cells after 2h of I-Ppo1 WT treatment. All scale bars = 10 µm. (D) Quantification of the experiment in (C) showing the number of BRCA1 (upper panel) and RAD51 (lower panel) nucleolar caps per cell (n = 100 cells). Graphs represent a single replicate (experiment was performed in duplicates) and bars represent mean. (E) Nucleolar EU incorporation in RPE1 ΔTP53 and RPE1 ΔTP53 ΔBRCA1 cells treated with I-Ppo1 WT or H98A for 2 hours. (F) Quantification of nucleolar EU incorporation after WT or H98A I-Ppo1 transfection in RPE1 ΔTP53 and RPE1 ΔTP53 ΔBRCA1 cells. Around 170 cells were considered per sample. Individual boxes represent the 25-75 percentile range with median and whiskers represent the 5-95 percentile range. Data points outside of this range are shown individually.
(A) Clonogenic survival assay of RPE1 WT, ΔMDC1 and H2AXS139A cells as well PRE1 ΔTP53 (Parental) and RPE1 ΔTP53 ΔBRCA1 cells after 2h of I-Ppo1 treatment. (B) Clonogenic survival analysis of I-Ppo1 WT treated RPE1 WT, ΔMDC1 and H2AXS139A or PRE1 ΔTP53 (Parental) and RPE1 ΔTP53 ΔBRCA1 cells. Each dot represents an independent replicate, black bars show means. (C) PLA assay showing the localization between EdU and γH2AX after H98A or WT I-Ppo1 transfection in U2OS or U2OS ΔMDC1 cells. All scale bars = 10 μm. (D) Quantification of the experiment in (C) showing the mean intensity of PLA signal within the nucleolar periphery U2OS or U2OS ΔMDC1 cells. Graph represents a pool of three independent experiments (n = 400 cells) and bars represent mean.
A proposed model of nucleolar response to DSBs.
