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. 2020 Nov 30;21(23):9130. doi: 10.3390/ijms21239130

Figure 4.

Figure 4

(A) IFM: DNA-SCARS identification. IFM micrographs of 53BP1 and PML staining of NT fibroblasts following 20 Gy (5 h, 24 h, and 2 weeks post-IR). Quantification of 53BP1-foci co-localizing with PML foci to detect acute DNA-repair foci and persistent DNA-SCARS in NT and KD fibroblasts. (B) IFM: DNA-SCARS accumulation and size. IFM micrographs of DNA-SCARS in non-IR controls, and 24 h and 2 weeks following 20 Gy in NT and KD cells. Quantification of DNA-SCARS in non-IR controls and 2 weeks after 20 Gy in NT and KD fibroblasts (upper panel). Area measurements of DNA-SCARS 24 h and 2 weeks following 20 Gy (lower panel). Data are presented as mean of three experiments ±SE. Significant statistical difference compared to non-irradiated controls (marked by asterisks alone) or between NT and KD cells (asterisks with square brackets): * p < 0.05; ** p < 0.01; *** p < 0.001. (C) TEM: H2A.J/53BP1 localization in DNA-SCARS. TEM micrographs show gold-labelled H2A.J (6-nm beads) and 53BP1 (10-nm beads) in NT and KD fibroblasts following 20 Gy (2 weeks post-IR), compared to non-IR controls. Red marked areas are shown with higher magnification; to aid visualization of gold-beads, green and red overlays were added, and bead clusters encircled (green: H2A.J only; red: 53BP1 only; yellow: co-localization of H2A.J and 53BP1).