Fig. 1

Nitric oxide preferentially induced cell death in AA breast cancer cells. AA and CA breast cancer cell lines were propagated in their respective media and treated with various concentrations of a) DETA-NONOate (DN)(0.01-0.3mM) for 48h. b) DETA-NONOate (0.5-1mM) for 48h c) DETA-NONOate (1mM) for 8-24h. Following these various treatments, the cells were harvested and their viability was determined by trypan blue exclusion method using hemocytometer for cell counting. d) Cells propagated on 8 well chamber slides were treated with DETA NONOate (1mM) for 24 and 48h. After these treatments the cells were fixed with 4% paraformaldehyde for 30 mins and DNA fragmentation determined by performing TUNEL assay. A representative picture of control and TUNEL positive MDA-MB-468 and MDA-MB-231 cells are shown. e) Quantitation of TUNEL positive cells was performed on images taken at 100 × magnification. Number of TUNEL positive cells/field (average of 3 fields) for each time points/cell line is shown. f) Cells treated with DETA-NONOate (1mM) for 48h were lysed in insect cell lysis buffer and 3μg of total cell lysates were subjected to caspase-3 activity assay using florescent substrates. Results from 3 different experiments were represented. g) Immunoblot analysis for heme oxygenase-1 (HO-1) was performed using 100μg of total cell lysates obtained from cells treated with DETA-NONOate (1mM) for various time points (0-24h). We used β-actin as housekeeping control. Two way ANOVA statistics was performed for A, B, C, E and F. Data are represented as mean ± SD with significant values presented as *p < 0.01, **p < 0.001, ***p < 0.0001