Regulation of Itch in chronic lymphocytic leukemia. (A) Western blotting of expression levels of protein Itch. Primary chronic lymphocytic leukemia (CLL) cells were incubated in presence of 10 nM LBH589 for the indicated times. Levels of actin were used as a loading control. Results from a representative experiment using CLL cells from 1 of the 12 analyzed patients are shown. There is a decrease in the 100 kDa band but with concomitant increase in 64 kDa cleaved product. See also Rossi et al³ . (B) Luciferase reporter assay of miR106b on 3′UTR-Itch in Saos-2 and HEK293 cells. The results were expressed as mean + SD from 3 independent experiments analyzed in triplicate. (C) Western blotting of endogenous levels of protein Itch in Saos-2 and HEK293 cells upon transfection. Cells were transfected either with pcDNA plasmids harboring the mature miR106b or a scrambled sequence, or with pre-miRs for miR106b or a scrambled sequence. Levels of actin were used as a loading control. MiR106b levels were assayed by qRT-PCR, to verify the transfection. (D) Expression levels of TAp73, DNp73, miR34a, and miR106b in primary CLL cells exposed for the indicated times to 10 nM LBH589 as analyzed by qRT-PCR. Expression levels relative to the untreated sample at the corresponding time points are shown. Expression of TAp73 and DNp73 was normalized to actin; expression of miR34a and miR106b was normalized to RNU6b. Statistical differences were determined by one-way analysis of variance (ANOVA) followed by Dunnet multiple comparison test, and the results were expressed as mean ± SEM from 12 independent CLL samples analyzed in triplicate.