Journal: Nature Communications

Article Title: Double-strand breaks in facultative heterochromatin require specific movements and chromatin changes for efficient repair

doi: 10.1038/s41467-024-53313-2

Figure Lengend Snippet: A Cell culture set up to analyze the dynamics of ATRIP localization upon DSB induction using 5Gy gamma-irradiation. Figure partly created in BioRender. Janssen, A. (2023) BioRender.com/o68s986 and Janssen, A. (2023) BioRender.com/v72e853. B Representative images of ATRIP-eYFP focus (green) dynamics within the ph-p-mCherry domain (magenta). Arrow indicates ATRIP-eYFP focus arising within ph-p-mCherry domain (0 min) and moving outside this domain (10 min). Scale bar = 2 μm. Dotted lines outline nuclei. C Quantification of ATRIP-eYFP (DSB) localization 10 min after ATRIP-eYFP appearance, 3 biological replicates per condition (yellow control dsRNA or dUtx dsRNA). Colors indicate localization of the ATRIP-eYFP focus (inside polycomb body [purple], outside [green], not detectable or resolved [gray]). p-value: 0.0490. D Relative dUtx expression level in Drosophila Kc cells normalized to an internal control gene (tubulin), determined using RT-qPCR. Bars indicate averages +SD of 3 biological replicates per condition. p-value: 0.0053. E Quantification of the number of cells with an ATRIP-eYFP focus inside ph-p-mCherry domain divided by the total number of cells expressing both ATRIP-eYFP and ph-p-mCherry. Each dot indicates the percentage of cells with ATRIP foci overlapping with a ph-p domain per acquired image (total of 20 images (control) and 18 images (dUtx dsRNA) were aquired during 3 biological replicates). Averages +SEM are shown. p-value: 0.0289. (ns) not significant, (*) p-value ≤ 0.05, (**) p-value ≤ 0.01, two-sided Chi-square test ( C ), two-sided paired t-test ( D ) and two-sided unpaired t-test ( E ). Source data are provided as Source Data file.

Article Snippet: To induce RNAi-mediated depletion of dUtx, cells were transfected with 5–10 ug dsRNA (TransIT-2020 reagent (Mirus)) and harvested or subjected to imaging 3 days later. dsRNA was generated using a MEGAScript T7 transcription kit (Life Technologies).

Techniques: Cell Culture, Irradiation, Control, Expressing, Quantitative RT-PCR

Journal: Nature Communications

Article Title: Double-strand breaks in facultative heterochromatin require specific movements and chromatin changes for efficient repair

doi: 10.1038/s41467-024-53313-2

Figure Lengend Snippet: A DR- white / hsp.I-SceI larvae with (+) or without (˗) dUtx RNAi were heat-shocked for one hour to induce I-SceI. Repair PCR products were Sanger sequenced 24 h after I-SceI induction and analyzed using the TIDE algorithm . Graph shows percentage of identified homologous recombination (HR) products. Bars indicate averages +SEM of n = 14 single larvae (biological replicates) per condition (except 3eu_1 , 3eu_2, 2fH_1 : n = 8, 3eu_1 (dUtx RNAi): n = 5, 3eu_2 (dUtx RNAi): n = 7, and 3fH_1 (dUtx RNAi): n = 12). p-values: 2fH_1 = 0.0122, 3fH_1 = 0.0005. B Representative image of a nucleus 60 min after 5 Gy irradiation immunostained for anti-yH2Av (yellow), anti-Rad51 (cyan) and anti-mCherry to visualize ph-p-mCherry (magenta). Zoom-in (below) shows a yH2Av focus colocalizing with Rad51, outside a php-mCherry domain. Scale bar = 1 μm. Dotted line outlines nucleus. C Quantification of the number of Rad51 foci per cell in the absence (control, yellow dsRNA, black line) and presence of dUtx dsRNA (orange line). Average number of Rad51 foci per cell is shown at indicated time points. Shade represents SEM of n = 4 (control) or n = 3 (dUtx dsRNA) biological replicates (with ≥10 cells per condition). p -value: 60 min = 0.0067. D Quantification of the localization of Rad51 60 minutes after 5 Gy IR either ‘inside’ a polycomb body or ‘outside’ a polycomb body. Dots represent n = 4 (control) or n = 3 (dUtx dsRNA) biological per condition (with ≥10 cells per condition). Straight line in the violin plot indicates the median and dotted lines indicate quartiles. p -value: outside = 0.0110. (ns) not significant, (*) p-value ≤ 0.05, (**) p-value ≤ 0.01, (***) p-value ≤ 0.001, two-sided unpaired t-test ( A , C , D ). Source data are provided as Source Data file.

Article Snippet: To induce RNAi-mediated depletion of dUtx, cells were transfected with 5–10 ug dsRNA (TransIT-2020 reagent (Mirus)) and harvested or subjected to imaging 3 days later. dsRNA was generated using a MEGAScript T7 transcription kit (Life Technologies).

Techniques: Homologous Recombination, Irradiation, Control