Research Paper Volume 12, Issue 13 pp 13594—13617

Figure 3. The expression of exogenous CREB sensitizes mouse lens epithelial cells to 40 mU GO-induced apoptosis (C, F). (A) Western blot analysis of the CREB levels in αTN4-1, pCI-αTN4-1, and pCI-CREB-αTN4-1 cells. (B) Semi-quantification of the western blot results in (A). (C and F) The αTN4-1, pCI-αTN4-1, and pCI-CREB-αTN4-1 cells were grown to 90% confluence. Then, 40 mU GO was added into the 3 types of cells, and the 3 types of cells were treated for indicated time. At the end of treatment, the cells were harvested for either live/dead assays (C), or for CellTiter-Glo® Luminescent Cell Viability Assay analysis [89] (F) to determine the rate of apoptosis. Note that pCI-CREB-αTN4-1 cells displayed the highest level of apoptosis (nearly 100%) in the 40mU glucose oxidase treatment (F). Green fluorescence represents live cells as detected by Calcein-AM, and red fluorescence detected by EthD-1 refers to dead cells. (D) Dynamic H₂O₂ concentration generated from 40mU glucose oxidase (GO) in the DMEM medium. (E) Dynamic changes of free thiol levels in mouse lens epithelial cells cultured in the DMEM medium under 40 mU GO treatment. All experiments were repeated three times. Error bar represents standard deviation, N=3. ** p<0.01.