Fig 2: Response over time to TBG-RNAi-CK2 treatment in PC3-LN4 xenograft tumors(A) Verification of nuclear and cytosol protein fractionation from tumor tissue is shown by comparative immunoblot analysis of CK2aa', lamin A/C, ERp72, and actin. T1, T2, and T3 labels indicate different tumors. Antibody sourcing information is listed in Materials and Methods. (B) Immunoblot analysis of fractionated PC3-LN4 tumor lysates following intravenous treatments of 0.01 mg/kg TBG-RNAi-CK2 or TBG-RNAi-F7 as indicated above the blots. The signals for three mice per group are shown, the proteins detected are indicated on the right, and the cell fraction and size markers are indicated on the left. Actin signal was used as the loading control. T1, T2, and T3 labels indicate different tumors within each treatment group. Antibody sourcing information is listed in Materials and Methods. (C) The percentage of Ki-67-positive tumor cells was determined from Ki-67-stained tumor sections. The percentage of Ki-67-positive cells on each day was expressed relative to day 5 (setting day 5 as 100%) and is shown graphically for each treatment. Mean and standard deviation are shown. Sample sizes: n = 6 for all groups except n = 7 for TBG-RNAi-F7 day 7.

Fig 3: Dose response to TBG-RNAi-CK2 and TBG-siCK2 treatment in PC3-LN4 and 22Rv1 xenograft tumors(A) The changes in PC3-LN4 tumor volumes relative to day 0 (day 10/day 0) are shown following drug treatments. Nanocapsule drugs and doses are indicated below the bars. Means ± standard deviations are presented and significance is indicated on the chart. The best response to both TBG-RNAi-CK2 and TBG-siCK2 treatments was observed at 0.01 mg/kg. Group sizes TBG-RNAi-CK2: 1 mg/kg n = 3; 0.1 mg/kg n = 4; 0.01 mg/kg n = 9, 0.001 mg/kg n = 8; 0.0001 mg/kg n = 8. TBG-RNAi-F7: 0.01 mg/kg n = 8. Groups sizes TBG-siCK2: 1 mg/kg n = 7; 0.1 mg/kg n = 8; 0.01 mg/kg n = 9. TBG-siCON1: 1 mg/kg n = 8. (B) The changes in 22Rv1 tumor volumes relative to day 0 are shown following drug treatments. Nanocapsule drugs and doses are indicated below the bars. Means ± standard deviations are presented. The best response to TBG-RNAi-CK2 treatment was observed at 0.1 mg/kg. Groups sizes TBG-RNAi-CK2: 1 mg/kg n = 9; 0.1 mg/kg n = 12; 0.01 mg/kg n = 8. TBG-RNAi-F7: 0.1 mg/kg n = 13. (C) The percent of Ki-67 positive cells for PC3-LN4 and 22Rv1 tumors are shown for the best response dose corresponding to 0.01 mg/kg TBG-RNAi-CK2 and TBG siCK2 for PC3-LN4 and 0.1 mg/kg for 22Rv1. The decrease in Ki-67 staining due to anti-CK2 nanocapsule treatment was from 3.6 to 4.6%. Nanocapsule drugs are indicated within the bars. Means ± standard deviations are presented. Sample sizes - PC3-LN4: TBG-RNAi-CK2 n = 6; TBG-RNAi-F7 n = 8; TBG-siCK2 n = 7; TBG-siCON1 n = 9. 22Rv1: TBG-RNAi-CK2 n =9; TBG-RNAi-F7 n = 9.

Fig 4: Blocking CK2 expression and activity reduces NF?B p65 protein levels and activation in PCa cells. (A) LNCaP cells were transfected with CK2aa’-targeted and control siRNAs. Cells were collected 48 and 72 h post-transfection, as labeled below the blots, for analysis by immunoblot. (B) C4-2B and 22Rv1 cells were treated with 20 µM TBB, 40 µM TBB or equivalent concentration of DMSO as indicated. Cells were collected at 24 h for analysis by immunoblot. Vertical lines indicate non-contiguous lanes. For all panels: proteins detected are indicated on the right side of blots and either actin or ß-tubulin were used as loading controls. Arrows indicate correct band. Means and 95% confidence intervals for values relative to siControl or DMSO are indicated below each blot.

Fig 5: Immunoblot analyses following small interfering RNA-mediated downregulation of CDK11 and CK2 in breast cancer cells. Immunoblot analysis of MDA-MB-231 and SUM-149 cell lysates following small interfering RNA (siRNA) transfection. Transfected siRNAs are indicated above the blots, proteins detected are indicated on the right side of the blots. CDK11p110, cyclin L1a, cyclin L2a, and CK2aa'ß lysates are 72 hours post transfection; caspase 3, Bcl-xL, and survivin lysates are 96 hours post transfection. Actin signal was used as the loading control. CDK, cyclin-dependent kinase; CK2, casein kinase 2.