C. S2 Fig: DNA damage and apoptosis-related chromatin compaction in HeLa cells after 24 h of mCherry-CdtB manifestation. A. Representative images of mCherry-HducCdtB localisation, H2AX immunofluorescence and DAPI staining from HeLa cells expressing WT or mutant (Hduc D273R or Ecol H153A) CdtB in fusion with mCherry. Immunostaining was performed 24 h after transfection. Cells with high manifestation (celebrities) of mutant mCherry-CdtB or with compacted chromatin (arrows) are indicated. Level pub: 20 m. B. Quantification of H2AX positive HeLa cells remaining untransfected (NT) or expressing mutant CdtB (HducCdtB D273R or EcolCdtB H153A). Immunostaining was performed 24 h after transfection. Results present the imply SD of at least three self-employed experiments; statistical variations were analysed between transfected and non-transfected conditions (not significant).(TIF) pone.0214313.s002.tif (791K) GUID:?2C63CBC4-702F-4A2F-9695-69859295FCCE S3 Esomeprazole sodium Fig: DNA damage induction in U2OS cells after CDT holotoxin treatment, mCherry-CdtB expression or purified CdtB transfection. A. Representative images of H2AX immunofluorescence and DAPI staining from U2OS cells treated with 20 ng/mL of WT or D273R HducCDT holotoxin or with 2.5 ng/mL of WT or H153A EcolCDT holotoxin for 24 h. Scale pub: 20 m. B. Quantification of H2AX positive U2OS cells remaining untreated (NT), treated with 20 ng/mL of Esomeprazole sodium WT or D273R HducCDT or with 2. 5 ng/mL of WT or H153A EcolCDT for 24 h, represented as the mean fluorescence intensity per cell (normalised to 1 1 for the untreated condition) or as the proportion Esomeprazole sodium of H2AX positive cells. Results present the imply SD of at least three self-employed experiments; statistical variations were analysed between treated and untreated conditions (** P < 0.01; **** P < 0.0001). C. Representative images of mCherry-HducCdtB localisation, H2AX immunofluorescence and DAPI staining from U2OS cells expressing WT or mutant CdtB (HducCdtB D273R or EcolCdtB H153A) Esomeprazole sodium in fusion with mCherry. Immunostaining was performed 11 h after transfection. Cells with high manifestation (celebrities) or low manifestation (arrows) of WT mCherry-HducCdtB are indicated. Level pub: 20 m. D. Quantification of H2AX positive U2OS cells remaining untransfected (NT), expressing WT or mutant CdtB (HducCdtB D273R or EcolCdtB H153A). Immunostaining was performed 11 h after transfection. Results present the imply SD of at least three self-employed experiments; statistical variations were analysed between transfected and non-transfected conditions (**** P < 0.0001). E. Representative images of H2AX immunofluorescence and DAPI staining from U2OS cells transfected with 120 nM of WT or mutant (Hduc D273R or Ecol H153A) CdtB for 14 h. Level pub: 20 m. F. Quantification of H2AX positive U2OS cells remaining untransfected (NT), transfected with 120 nM of WT or mutant (Hduc D273R or Ecol H153A) CdtB for 14 h, represented as the mean fluorescence intensity per cell (normalised to 1 1 for the untreated condition) or as the proportion of H2AX positive cells. Results present the imply SD of at least three self-employed experiments; statistical variations were analysed between treated and untreated Esomeprazole sodium conditions (* P < 0.05; **** P < 0.0001) or between HducCdtB and EcolCdtB (## P < 0.01; #### P < 0.0001).(TIF) pone.0214313.s003.tif (2.4M) GUID:?328CA011-F1AA-4CE0-BD93-FB784F22946B S4 Fig: Dose-response analysis of the plasmid digestion assay with EcolCdtB and HducCdtB purified less than native conditions. A. Dose-response analysis of the plasmid digestion assay in presence of WT or D273R HducCdtB. Agarose gel electrophoresis and quantification of supercoiled plasmid (250 ng) incubated with the indicated concentrations of WT or D273R HducCdtB for 10 min. M: molecular excess weight marker. B. CdtB concentration effect on the plasmid digestion assay in presence of WT or H153A EcolCdtB. Agarose gel electrophoresis and quantification of supercoiled plasmid (250 ng) incubated with the indicated concentrations of WT or D273R HducCdtB for 10 min. Rabbit polyclonal to FBXO10 M: molecular excess weight marker. Arrows show plasmid conformation, either.