Figure 1. Localization of Grx1 in cytosol and generation of ROS following its knockdown in Neuro-2a cells.

Cells were loaded with MitoTracker Deep Red 633 (500 nM) for 45 min before fixation and subsequent immunostaining for Grx1 or Grx2. Images were captured using LSM510 META in confocal microscope. (A) Grx1 (green) does not colocalize with MitoTracker Deep Red 633 (red) in Neuro-2a cells indicating its cytosolic localization, whereas mitochondrial glutaredoxin 2 (green; Grx2) colocalizes with MitoTracker Deep Red. Bar represents 10 µm. (B) Quantitation of knockdown of Grx1 as assessed by qRT-PCR, no change observed in Grx2 mRNA levels. Data is represented as mean±SEM from 3 independent experiments. Asterisks indicate values significantly different from controls (p<0.01). (C) Immunoblots depicting Grx1 protein levels in control (con) and knockdown using shRNA (sh) in Neuro-2a cells and densitometric quantitation of immunoblot after normalization with β-tubulin. Data is represented as mean±SEM from 3 independent experiments. Asterisks indicate values significantly different from controls (p<0.01). (D) Immunostaining for Grx1 in Neuro-2a cells transfected with empty vector or shRNA to Grx1. Bar represents 100 µM. (E) Downregulation of Grx1 using shRNA in Neuro-2a cells enhances ROS production as seen by increased H₂DCFDA staining. Bar represents 100 µm.