Figure 2.

Activation of CK2 reduces number of PML-NBs. (a) Immuno-fluorescence detection of PML-NBs: PC3 cells were treated with d—Sorbitol and TBCA (prior harvesting) simultaneously with CHX. Cells were processed appropriately for immuno-fluorescence imaging and were observed under microscope. (b) Quantification of average number of PML-NBs. PC3 cells being treated with either d—Sorbitol or TBCA in combination with CHX. PML-NBs were counted from 480 cells (nuclei) out of 80 randomly chosen fields (16 fields from each experiment; number of biologically repeated experiments being five). Bars represent mean (±) S.D. of the five independent biological repeats with the *P=0.031 level of significance. (c) Level of PML protein is increased upon inhibition of CK2 activity. PC3 cells were treated either with d—Sorbitol or TBCA and lysates were separated on SDS-PAGE and probed with mentioned antibodies. (d) Activation of CK2 decreases PML protein stability. PC3 cells were treated with d—Sorbitol along with CHX. Cells were harvested, and lysates were subjected to IB and probed with the indicated antibodies. (e) PML protein level increases with increasing duration of CK2 inhibition. PC3 cells were treated either with TBCA or with d—Sorbitol. Lysates prepared from the treated cells were immunoblotted with anti-PML antibody. (f) Exogenously overexpressed CK2 decreases PML protein level by promoting proteasomal degradation. PC3 cells were transfected with either wild-type (WT) CK2α alone or followed by MG132 treatment (after 32 h of post-transfection). Lysates were prepared and subjected to IB analyses with respective antibodies as mentioned. Cells treated with TBCA served as positive control. Control cells had appropriate empty vector transfection along with DMSO treatment at the required quantity. In all the cases, bars represent (±) S.D. of three independent biological repeats at the mentioned level of significances. Cells treated with DMSO-water (in a, c, d) or water only (in e) were used as respective controls