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. 2019 Mar 28;14(3):e0214313. doi: 10.1371/journal.pone.0214313

Fig 4. DNA damage induction in HeLa cells transfected with purified CdtB.

Fig 4

A. Representative images of γH2AX immunofluorescence and DAPI staining from HeLa cells transfected with 50 nM of HducCdtB WT (fractions C2, C11, D4, E5) or D273R (fraction D2) after SEC for 24 h. Scale bar: 20 μm. B. Representative images of γH2AX immunofluorescence and DAPI staining from HeLa cells transfected with 50 nM of WT or D273R HducCdtB before SEC, with 200 nM of DNase I or with 120 nM of HducCdtC for 24 h. Scale bar: 20 μm. C. Representative images of γH2AX immunofluorescence and DAPI staining from HeLa cells transfected with 120 nM of EcolCdtB WT or H153A for 14 h. Scale bar: 20 μm. D. Representative images of γH2AX and 53BP1 immunofluorescence and DAPI staining from HeLa cells transfected with 5 nM of WT or mutant (Hduc D273R or Ecol H153A) CdtB for 14 h. Scale bar: 20 μm. E. Dose-response analysis of γH2AX induction after CdtB transfection. Quantification of γH2AX signal in HeLa cells left untransfected (NT) or transfected with the indicated concentration of WT or mutant (Hduc D273R or Ecol H153A) CdtB or negative controls (HducCdtC or DNase I) for 14 h, represented as the mean fluorescence intensity per cell (normalised to 1 for the untreated condition) or as the proportion of γH2AX positive cells. Results present the mean ± SD of at least three independent experiments. Statistical differences were analysed between transfected and non-transfected conditions for each CdtB concentration (* P < 0.05; **** P < 0.0001) or between HducCdtB and EcolCdtB (# P < 0.05; ## P < 0.01; #### P < 0.0001). F. Kinetics of γH2AX induction after CdtB transfection. Quantification of γH2AX signal in HeLa cells left untransfected (NT) or transfected with 120 nM of WT EcolCdtB or HducCdtB for the indicated time, represented as the mean fluorescence intensity per cell (normalised to 1 for the untreated condition) or as the proportion of γH2AX positive cells. Results present the mean ± SD of at least three independent experiments; statistical differences were analysed between transfected and non-transfected conditions for each time point (* P < 0.05; ** P < 0.01; ***P < 0.001; **** P < 0.0001) or between HducCdtB and EcolCdtB (# P < 0.05; ## P < 0.01; ### P < 0.001; #### P < 0.0001).