Figure 4

Vor-resistant cells efficiently sequester ROS and possess elevated catalase levels. (a) Relative MFI (versus U937 veh.) of DCFDA fluorescence after vehicle (DMSO), and 6, 12, and 18 h Vor treatment in U937, and B8 chronically maintained in Vor (2 μM). Graph represents means±S.E.M. (one-way analysis of variance, Tukey) of N=2 (technical triplicate). (b) Immunoblots of samples from a, probing CL-PARP, HO-1, γH2AX, and β-actin (loading control). (c) H₂O₂ decomposition assay. Relative (versus U937 veh.) changes in absorbance at 240 nm were monitored in vehicle, Vor-treated (2 μM) U937 cells for the indicated times, and B8 cells (2 μM Vor). Moles of H₂O₂ consumed was calculated via Beer’s law (see Materials and Methods). Graph represents means±S.E.M. (one-way analysis of variance, Tukey) of N=3 (technical triplicate). (d) PI-stained (sub G₁ population) U937 and B8 cells treated with vehicle, and 0.25 and 0.50 mM H₂O₂ for 24 h. Graph represents means±S.E.M. (one-way analysis of variance, Tukey) of N=3 (technical triplicate). (e) RT-qPCR analyses of CAT versus 36B4 in vehicle, Vor-treated (2 μM) U937 for the indicated times, and B8 cells chronically in Vor. Graph represents means±S.E.M. (one-way analysis of variance, Tukey) of N=5 (technical triplicate). (f) Immunoblots of catalase and β-actin (loading control) corresponding to conditions in (e). (g) Immunofluorescence staining of catalase (TO-PRO-3 nuclear stain), comparing vehicle and Vor-treated U937 cells, and B8 cells. Scale bar represents 10 μm. For all statistical tests ***P<0.001