Journal: FEBS Open Bio

Article Title: Myeloperoxidase inhibition protects bone marrow mononuclear cells from DNA damage induced by the TOP 2 poison anti‐cancer drug etoposide

doi: 10.1002/2211-5463.13799

Figure Lengend Snippet: PF1355 reduces the level of TOP2B‐DNA covalent complexes induced by etoposide in expanded CD34 + cells. (A) Cells were pre‐treated with PF1355 (10 μ m ) for 4 h before adding etoposide (100 μ m ). Cells were incubated with etoposide for 1 h and TOP2B‐DNA covalent complexes were quantified by TARDIS analysis. Integrated fluorescence values were determined per nucleus (at least 500 nuclei per treatment per replicate experiment). Data in the left panel represent the individual integrated fluorescence values per cell for four biological replicates. From these, median values were obtained for each treatment and means of the medians were calculated from replicates and plotted on the right expressed as a percentage of the mean value obtained with 100 μ m etoposide in the absence of PF1355 ± SEM. Significance testing was performed by unpaired t ‐test (** P < 0.01). (B) Standard immunofluorescence analysis of representative samples used for (A) demonstrating the broad expression of TOP2B and MPO. (C) Representative image from the TARDIS data enumerated in (A) and (B). CD34 + BMCs were cultured/expanded in SFEM + CC100 for 16 days prior to drug treatment. Scale bar = 50 μm.

Article Snippet: Thawed cells were expanded in Stemspan SFEM II medium with CC100 cytokine cocktail (Stemcell Technologies).

Techniques: Incubation, Fluorescence, Immunofluorescence, Expressing, Cell Culture

Journal: FEBS Open Bio

Article Title: Myeloperoxidase inhibition protects bone marrow mononuclear cells from DNA damage induced by the TOP 2 poison anti‐cancer drug etoposide

doi: 10.1002/2211-5463.13799

Figure Lengend Snippet: PF1355 suppresses etoposide‐induced DNA damage in ex vivo expanded CD34 + BM‐MNCs. (A) Expanded CD34 + cells were treated with the drug combinations shown, PF1355 (10 μ m ) pre‐treatment was for 4 h before adding etoposide. Cells were incubated with etoposide for 1 h, before attaching to poly‐ l ‐lysine‐coated coverslips and immunofluorescent staining for γH2AX. (B) Cells were treated as indicated, attached to poly‐lysine‐coated coverslips and H2AX phosphorylation was quantified by immunofluorescence. Data shown is the mean ± SEM of the median integrated fluorescence per nucleus obtained from four replicas. Statistical testing was by 1‐way ANOVA. (C) Violin plots illustrating the distribution of integrated fluorescence per nucleus values from one of the replicas employed in (B). (D) Analysis of γH2AX foci induced by etoposide and the effect on this of PF1355. Images were captured using a 40× objective and nuclei were placed into bins based on the number of γH2AX foci present. Data are the mean values obtained from three replicas ± SEM. Significance testing was performed using 2‐way ANOVA (Tukey; * P < 0.05, *** P < 0.001, **** P < 0.0001). CD34 + BMCs were cultured/expanded for 11 days in culture SFEM+CC100 prior to drug treatment. Scale bar = 50 μm.

Article Snippet: Thawed cells were expanded in Stemspan SFEM II medium with CC100 cytokine cocktail (Stemcell Technologies).

Techniques: Ex Vivo, Incubation, Staining, Phospho-proteomics, Immunofluorescence, Fluorescence, Cell Culture