Figure 3. Increased expression of myocilin after transfection. RT- PCR and western blot show that transfection of WT or Pro370Leu (P370L) mutant myocilin increase both myocilin mRNA and protein levels in TM cells after 48 h. GAPDH was used as the internal loading control in RT–PCR while β-actin and cytochrome C oxidase (COX IV) were used as internal loading controls in western blot analysis for total cell lysate and mitochondrial lysate, respectively. Confocal images show myocilin co-localized with mitochondria. After transfection for 8 h, non-fixed TM cells were immunolabeled with MitoTracker Red, a mitochondria specific dye. Confocal microscopic analyses show that both pIRES-EGFP-WT myocilin (WT) and pIRES-EGFP-Pro370Leu mutant myocilin (P370L) transfected cells have overlapping EGFP staining (green fluorescence) and mitochondria staining (red fluorescence). However, the green fluorescence from pIRES-EGFP (Mock) is diffusive within the cells with no overlapping with mitochondrial staining. Our result indicates co-localization of myocilin and mitochondria in TM cells. Scale bar=30 μm.