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. 2013 Mar 28;8(3):e59505. doi: 10.1371/journal.pone.0059505

Figure 3. Conditional knockout of LIG3 reveals the function of LIG1 in the processing of IR-induced DSBs.

Figure 3

(A) Kinetics of DSB processing in the indicated mutants after treatment with 4HT for the indicated periods of time. Other details are as in Fig. 1B. Results from three independent experiments with 3 samples each were used to calculate the indicated means and standard errors. (B) LIG3 mRNA levels measured by real-time PCR in wt and LIG3−/2loxP cells after different incubation times with 4HT. The mRNA level measured in wt cells was set to 100%. (C) Western blot analysis of LIG3 protein in LIG3−/2loxP cells after treatment with 4HT for the indicated periods of time. A mouse monoclonal antibody against human LIG3 (clone 1F3) that recognizes the chicken LIG3 was used. GAPDH is a loading control. (D) Apoptotic index measured by microscopically scoring nuclear fragmentation and pycnosis in wt and LIG3−/2loxP cells at various times after treatment with 4HT. Results from two independent experiments in each of which 1000 cells were scored were used to calculate the indicated means and standard errors. (E) As in A for the indicated mutants. (F) Representative cell-cycle distribution histograms obtained by flow cytometry in wt, LIG4−/− and LIG3−/2loxPLIG4−/− cells treated with 4HT for 3.5 days before and after enrichment by centrifugal elutriation in G2 phase of the cell cycle. (G) Kinetics of DSB processing in cells enriched by centrifugal elutriation in the G2 phase of the cell cycle as shown in F.