TXNIP Links Innate Host Defense Mechanisms to Oxidative Stress and Inflammation in Retinal Muller Glia under Chronic Hyperglycemia: Implications for Diabetic Retinopathy
Figure 9
TXNIP knockdown by siRNA blocks HG-mediated ATP reduction, ROS generation, and LC3B expression in rMC1. (a) Seventy to eighty percent confluent rMC1 cells were transfected transiently with an scramble siRNA (scrRNA, control) or with TXNIP mRNA-targeted siRNAs-siTXNIP1 and siTXNIP3 (20 nM each) using HiPerfect transfection reagent in six-well plates or 60 mm culture plates in duplicates. After 6 h, complete media containing 5% serum were replaced and kept for 24 h. Media was subsequently changed to low serum media (0.2% serum) for 48 h. Afterwards, HG was added for 4 h and TXNIP amount was measured by Western blotting. We observe that siTXNIP3 gives a consistent suppression of TXNIP (~70%) and used in further studies. A representative blot of is shown here. Actin was used as a control for protein loading and siRNA specificity. (b) rMC1 cells were transiently transfected in 24-well plates with siTXNIP3 for 48 h and then HG was added for 72 h in low serum media. Intracellular ATP concentration was measured and normalized to protein concentration (RLUs/g protein). HG reduces ATP level in scrRNA-treated rMC1 cells while siTXNIP3 transfection blunted HG effects. (c) Similar to ATP determination, we also measured ROS levels after 72 h of HG exposure in both scrRNA and siTXNIP3-transfected rMC1 with CM-H2DCFDA. HG increases ROS levels in scrRNA cells (, ) versus LG but this effect was not observed in siTXNIP3 cells. (d) LC3B staining under HG exposure for 5 days is also reduced in siTXNIP3-transfected cells when compared with the scrRNA cells under similar duration of HG exposure (right panels). Under LG, LC3B staining is minimal in both scrRNA and siTXNIP3-treated rMC1 cells after 5 days. A representative of is shown here.
