Figure 5

PML/HSP70 co-aggregation is dependent upon ROS and is not maintained in puromycin resistant selected cells. (A) Low passage (<10 passages) and high passage U2OS cells were incubated with H2DCFDA and analyzed by flow cytometry to detect ROS levels. Representative flow cytometry histogram displaying levels of fluorescent H2DCFDA in low passage versus high passage U2OS cells. (B) U2OS cells on glass coverslips were transiently transfected with pBABE Puro or pEGFP Neo. ROS scavengers, catalase (CAT; 50 μg/mL) or N acetyl cysteine (NAC; 10 mM), were added to media of transfected cells 15 h after transfection. Transfected cells were immunostained for HSP70 and PML 48 h after transfection. Cells displaying aggregation of PML/HSP70 were quantified and expressed as a percentage of EGFP expressing cells. Data represents the average ± SEM from 3 independent experiments. Asterisk represents p-value < 0.01 compared to cells without ROS scavengers (Student T Test). (C) Representative images of puromycin selected cells demonstrating the absence of PML/HSP70 aggregation. U2OS cells were transfected with pBABE puro and selected in puromycin as described in materials and methods and then immunostained for HSP70 (green) and PML (red). PML and HSP70 antibodies were detected using a rhodamine-x conjugated goat anti mouse IgG and Alexa 488 goat anti rabbit IgG respectively. Cell nuclei were counterstained with DAPI (blue). Images were captured at 100× magnification.