Figure 4. Pro370Leu mutant myocilin induces ROS generation and increases calcium levels in the cytoplasm and mitochondria in TM cells as estimated using flow cytometry. A: The oxidation sensitive fluorescence dye, H₂-DCF-DA, was used to measure the ROS levels in TM cells. TM cells transfected with Pro370Leu (P370L) mutant myocilin transfection show 2.05 fold (±0.94) stronger H₂-DCF-DA fluorescent intensity than those transfected with WT myocilin (WT), indicating an increase in ROS generation after Pro370Leu mutant myocilin transfection compared to WT myocilin transfection. B,C: The [Ca²⁺]_c and [Ca²⁺]_m indicators, fluo-3/AM and Rhod-2, were used to illustrated the free Ca²⁺ levels in the cytoplasm and mitochondria, respectively. There was a 3.17 fold (±0.64) increase in [Ca²⁺]_c and 1.92 fold (±0.74) increase in [Ca²⁺]_m after Pro370Leu mutant myocilin (P370L) transfection compared to WT myocilin transfection (WT). Data are expressed as fold changes in fluorescent levels of transfected TM cells to non-transfected TM cells (Control). Results are expressed as the mean±SE of three repeated experiments done in triplicate. An asterisk indicates a significant difference from non-transfected cells at p<0.05.