Figure 6. Knockdown of USP9X in DAOY MB.

(A) Western blot analysis to verify the knockdown of USP9X in DAOY MB cells following infection with lentiviruses to introduce constitutively active shRNA against USP9X transcripts. Nuclear and cytoplasmic protein fractions were prepared 4 days after infecting cells with lentiviruses. USP9X levels are quantified, and levels in the Scrambled control are set to 1.00. (B) Cell growth was examined in triplicate by MTT assay 6 days after being plated at 10⁴ cells per well of a 12-well plate. The data shown are averages relative to the Scramble control. Error bars represent standard deviation. P values were determined by student t-test and found to be <.01 for both USP9X shRNA 1, 2 and 3. (C) Photomicrographs of DAOY MB cells following knockdown of USP9X using lentiviral delivered shRNA constructs against USP9X transcripts. On day 0, cells were infected with USP9X shRNA lentiviruses. Beginning on day 1, infected cells were selected using puromycin for 48 hours. On day 4, cells were passaged into fresh culture flasks (5×10⁵ cells per flask) and photographed on day 8 post-infection. (D) Western blot analysis of Mcl1, and β-catenin – targets of USP9X – in DAOY MB extracts used in Figure 6A.