Research Paper Volume 12, Issue 13 pp 13594—13617

Figure 4. Comparative transcriptome analysis. (A–C) Both pCI-αTN4-1 and pCI-CREB-αTN4-1 cells were grown to 90% confluence and then harvested for RNAseq analysis. The gene expression patterns between vector-transfected cells and wild type CREB-transfected cells were compared (SRA accession: PRJNA566306). Compared to pCI-vector, expression of the exogenous WT-CREB caused changes in the expression patterns of 1916 genes, 872 genes were upregulated and 1044 genes were downregulated (A). (B) Hierarchical cluster analysis of apoptosis-associated genes. (C) The expression levels of the anti-apoptotic gene αB-crystallin in pCI-αTN4-1 and pCI-CREB-αTN4-1 cells (B) were further verified by qRT-PCR. Note that the expression of anti-apoptotic gene coding for αB-crystallin was significantly downregulated in pCI-CREB-αTN4-1 cell. (D–F) CREB downregulates expression of αB-crystallin during H₂O₂-induced apoptosis of CREB-expressing cells. (D) Apoptosis rate changes in αTN4-1, pCI-αTN4-1 and pCI-CREB-αTN4-1 cells under treatment of 40 mU GO from 0 to 6 hours were measured by CellTiter-Glo® Luminescent Cell Viability Assay analysis [89]. (E) Western blot analysis of the expression levels of αB-crystallin in αTN4-1, pCI-αTN4-1 and pCI-CREB-αTN4-1 cells under 40 mU GO. Note that the expression level of αB-crystallin was significantly downregulated in pCI-CREB-αTN4-1 cell. In addition, 40 mU GO treatment downregulated expression level of αB-crystallin in αTN4-1, pCI-αTN4-1 and pCI-CREB-αTN4-1 cells. (F) Semi-quantification of the western blot results in E. All experiments were repeated three times. Error bar represents standard deviation, N=3. ** p<0.01.