Fig. 3

Differential DDR markers in early and late phases of micronuclei. a: DAPI staining showing the dynamic changes of IR-induced MN in OVCAR-8.Early MN (white arrows) exhibited dense and compact DAPI staining while turned into loose structures with weaken DAPI staining upon transforming into late stages (red arrows). b: Counting for late stage MN after 2-Gy irradiation at indicated time points. n = 3 biological repeats. Error bars = s.d. c-e: Indirect immunofluorescent staining for γH2AX (c) or 53BP1 (d) IRIF and quantifications (e) in early and late phases of MN. OVACAR-8TR cells were fixed and stained 36 h after IR treatments. Note that the lack of γH2AX foci and enriched 53BP1 foci in late stages. White arrows: early-stage MN; Red arrows: late-stage MN. f: IF staining of γH2AX and 53BP1 foci in post-IR mitotic cells, showing the retaining of γH2AX foci and exclusion of 53BP1 signal from chromosomes. g: Lack of γH2AX and 53BP1 co-localization in micronuclei compartments. Left: representative images showing strong γH2AX signals in early stages and 53BP1 foci in late stages. Right: calculation for percentage of γH2AX/53BP1 double positive foci in nuclear and MN compartments. Majority of these foci were overlapping in nuclei while co-localization events were rare in MN. n = 3 biological repeats. Error bars = s.d. ***P < 0.001 (t-test). Hh: Counting of γH2AX and 53BP1 foci in untreated (spontaneous) and IR-induced MN