Figure 2.

Aki1 maintains the viability of DMM cells through CREB1 activation. A, human kinase phosphorylation array analysis in 211H cells with scramble control siRNA (a) or Aki1 siRNA (b) after 48 hours incubation. B, total and phosphorylation of CREB expressions in four DMM (211H, H290, H2052, and Y-meso14) and mesothelial (Met-5A) cells. Cells were lysed and the indicated proteins were detected by Western blotting. C, luciferase activity assay against CREB1. Cells were stably transduced to express wild-type CRE reporters, which binds to CREB1 as a certain DNA sequences, and ratios between the two were compared after RNAi transfection of Aki1 or scramble control; *, P < 0.05 versus scramble control by one-way ANOVA. D, cells were treated with scramble control, Aki1, or CREB1 siRNA. After 24 hours incubation, cells were lysed and the indicated proteins were detected by Western blotting analysis. E, cells were treated with Aki1, CREB1 (#1, #2), or scramble control siRNA. After 24, 48, 72, and 96 hours incubation, cell viability was determined by MTT assay. F, pCF plasmid–expressing empty vector (Vector), FLAG-tagged human CREB (CREB-WT), or FLAG-tagged human CREB-kinase dead–mutated (CREB-KD) clones were transfected into 211H cells. They were lysed and the indicated proteins were detected by Western blotting analysis. G, total and phosphorylation of CREB1 expressions in 211H cells with transfection of vector, CREB-WT, or CREB-KD clones were treated with Aki1 or scramble control siRNA. After 24 hours incubation, cells were lysed and the indicated proteins were detected by Western blotting. H, 211H cells with transfection of vector, CREB-WT, or CREB-KD clones were treated with Aki1 or scramble control siRNA. After 72 hours incubation, cell viability was determined by MTT assay; *, P < 0.05 versus cells with vector or CREB-KD clones treated with Aki1 siRNA by one-way ANOVA.