Figure 2

Ormeloxifene induces apoptosis and arrests cell cycle of cervical cancer cells. Ormeloxifene decreases mitochondrial membrane potential (MMP). (A) Cells were stained with TMRE dye for 20 mins at 37 °C and next treated with ormeloxifene for 24 hours to detect the healthy mitochondria. Representation of qualitative images showed decreased TMRE stain signifying reduced MMP, images were taken at 200X. (B) Flow cytometry results also showed a reduction in MMP (decreased TMRE fluorescence level). Results were normalized to the ETOH control. Error bars show SEM, n = 3. *p < 0.05. Ormeloxifene induces apoptosis. (C) Cells were treated with ormeloxifene for 24 hours and analyzed by flow cytometry using Annexin V and 7AAD dyes. (D) Graphical representation of flow cytometry data for Annexin V positive cells (early apoptosis). Error bars show SEM, n = 3. *p < 0.05. (E) Generation of reactive oxygen species (ROS). Cells were treated with 25 µM ormeloxifene and stained with DCFH-DA dye. Flow cytometry data represented an elevated levels of DCFH-DA dye which denotes generation of ROS. Error bars show SEM, n = 3. *p < 0.05. (F) Cell cycle was arrested at G1-S transition. Cells were treated with ormeloxifene for 24 hours, stained with PI dye and analyzed by flow cytometer and ModFit software for cell cycle analysis. ± shows SEM, n = 3. *p < 0.05.