A. Simultaneous cJUN and JUNB silencing was performed using siRNA for both genes and was confirmed by Western blot analysis. B. Combined JUNB and cJUN knockdown had an additive effect on the reduction of AKT1 promoter activity resulting in decreased AKT1 levels (not shown). C and D. In two NPM-ALK(+) ALCL cell lines, Karpas 299 (C) and SR-786 (D), CJUN/JUNB silencing attenuated AKT1, AKT2 and AKT3 protein levels. Moreover, phosphorylation of GSK3 and AKT1 kinase activation (as shown here by T308 and S473 phosphorylation of AKT1) were substantially lowered. Of note, transient JUNB gene inhibition, increased AKT2 protein levels in both cell lines. E and F. Finally, silencing both JUNB and CJUN led to a substantial decrease in AKT3 and (E) AKT2 (F) mRNA expression levels. qRT-PCR was performed in triplicates, and the values were normalized against the expression levels of GAPDH.
p<0,05, **, p<0,01, ***, p<0,001 (Student’s t-test).