Research Paper Volume 12, Issue 13 pp 13594—13617

Figure 6. Exogenous human αB-crystallin restores the ability of pCI-CREB-αTN4-1 cells against hydrogen peroxide-induced apoptosis. (A) The pCI-CREB-αTN4-1 cells were either untransfected (A-a), or transfected with pEGFPC3 vector (A-b), or pEGFPC3-HαB (A-c) transiently. Transfection was confirmed by fluorescence microscopy. The pEGFPC3 vector-transfected pCI-CREB-αTN4-1 cells displayed homogenous distribution of green fluorescence protein in the whole cells (A-b). In contrast, in the pEGFPC3-HαB-transfected pCI-CREB-αTN4-1 cells, the green fluorescence fusion protein was largely restricted in the cytoplasm (A-c). (B) Western blot analysis of the expression level of endogenous αB-crystallin and GFP-αB fusion protein in pCI-CREB-αTN4-1 cells (a), pCI-CREB-αTN4-1/pEGFPC3-αTN4-1 cells (b) and pCI-CREB-αTN4-1/pEGFPC3-HαB-αTN4-1 cells (c) detected with anti-αB antibody. (C) Western blot analysis of the expression level of GFP and GFP-αB fusion protein in pCI-CREB-αTN4-1 cells (a), pCI-CREB-αTN4-1/pEGFPC3-αTN4-1 cells (b) and pCI-CREB-αTN4-1/pEGFPC3-HαB-αTN4-1 cells (c) detected with anti-EGFP antibody. (D) After treatment by 40 mU GO for 6 hours, apoptosis in the 3 types of cells as indicated were analyzed. Note that pCI-CREB-αTN4-1/pEGFPC3-HαB-αTN4-1 cells expressing exogenous HαB displayed over 50% less apoptosis than pCI-CREB-αTN4-1 cells and pCI-CREB-αTN4-1/pEGFPC3-αTN4-1 cells. All experiments were repeated three times. Error bar represents standard deviation, N=3. * p<0.05; NS, statistically not significant.