Fig 1: Silencing CKB promotes prostate cancer cell migration and focus formation in vitro, as well as primary tumor growth and lung metastasis in vivo. (A) Cell migration as determined by Boyden chamber assay for PC3 and DU145 cells stably expressing control or CKB shRNA. Representative pictures from triplicate experiments are shown. (B) Boyden chamber cell migration assay for PC3 and DU145 cells transfected with control siRNA or CKB siRNA. Representative pictures from triplicate experiments are shown. Quantifications are in Supplementary Figure S3B. (C) Focus formation of PC3 cells stably expressing control or CKB shRNA. Representative pictures from duplicate experiments are shown. Additional pictures are in Figure S3C. (D) Relative cell growth rate of PC3 cells expressing control shRNA (shScram) or shCKB-1 in culture medium supplied with low % of FBS (1.25%), at day 1, 3, 8 and 10 (quadruplicates). Relative fold changes to day 1 were calculated and plotted as Means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 from comparing shScram with shCKB-1 cells at each time points (quadruplicates). (E) PC3 cells labelled with luciferase and expressing either shScram or shCKB-1 were implanted into prostates of NOD/SCID mice (shScram n = 5, shCKB-1 n = 4 mice). Fold changes of bioluminescence readings were calculated and plotted as Means ± SD (middle). Representative bioluminescence images of mice before sacrifice at day 85 (left) and ex vivo images for lung metastasis after sacrifice (right) are shown. (F) DU145 cells labelled with luciferase and expressing either shScram or shCKB-1 were implanted subcutaneously in NOD/SCID mice (shScram n = 8, shCKB-1 n = 8 mice). Fold changes of bioluminescence readings were calculated and plotted as Means ± SD (middle). Representative bioluminescence images of mice before sacrifice at day 35 (left) and ex vivo images for lung metastasis after sacrifice (right) are shown. *P < 0.05, **P < 0.01 comparing shScram with shCKB-1 group at the indicated time points (E and F).
Fig 2: Silencing CKB induces EMT markers in prostate cancer cells. (A) qPCR analysis of PC3 cells stably expressing scramble control shRNA or 2 independent CKB shRNAs (shCKB-1 and shCKB-2). Relative fold change was calculated and plotted as means ± SD. *P < 0.05 comparing shScram to either shCKB group. (B) Immunoblotting of whole cell lysates of PC3 cells stably expressing scramble control shRNA or CKB shRNA. (C) Immunoblotting in DU145 and VCaP cells expressing control shScram or shCKB. (D) Immunoblotting of whole cell lysates of DU145 cells transfected with control siRNA or CKB siRNA. (E) Immunoblotting of PC3 parental cells (WT) and 4 pools of PC3 cells transfected with 4 different Cas9-gRNA plasmids targeting CKB. These PCR and immunoblotting experiments have been repeated 3 times with comparable results, and so 1 representative experiment is presented.
Fig 3: CKB interacts with AKT and inhibits AKT activation. (A) CKB and AKT proteins could reciprocally co-immunoprecipitate (co-IP) each other from LNCaP cells. (B) AKT protein was immunoprecipitated by CKB antibody in a mixture of recombinant His-tagged AKT and His-tagged CKB proteins. (C) PC3-GFP and PC3-CKB cells were treated with or without 100ng/ml EGF for 5 min. Endogenous AKT was immunoprecipated from these 2 cell lines using AKT Ab, followed by immunoblotting for Rictor, mTOR and AKT (top). Conversely, endogenous Rictor was immunoprecipated using Rictor Ab, then immunoblot for AKT, mTOR and Rictor (middle). Immunoblotting of the input whole cell lysates was shown in bottom. Fold changes of Rictor and AKT protein levels in the IP samples are plotted, relative to the sample without EGF and without CKB overexpression. The quantification is based on measurements from 2 independent experiments, using ImageJ software.
Fig 4: CKB silencing induces AKT-S473 phosphorylation, consistent with their correlation in patient samples. (A) Whole cell lysate from PC3 cells expressing either shScram or shCKB-1 was tested on a human phospho-kinase array (R&D Systems). Red circles show duplicate spots with signals from p-S473-AKT antibody. (B) Immunoblotting for p-S473-AKT, CKB and Beta Actin in DU145 cells expressing shScram or shCKB-1 (left), and PC3 cells carrying empty vector control (EV, Ctrl) or CKB cDNA (right). (C) Immunoblotting for E-Cadherin, Vimentin, Slug, Twist, p-S473-AKT, AKT and beta-Actin in PC3 parental (WT) and CKB knockout cells. (D) Immunohistochemistry analysis of p-S473-AKT in PC3-shScramble and PC3-shCKB xenografts. (E) p-S473-AKT levels in TCGA primary prostate tumors, as analyzed by RPPA (accessed through cBioportal), comparing samples with low CKB mRNA expression (Z-score <-0.5) with all other samples. The p-S473-AKT plot and P value (P < 0.01) were obtained from cBioPortal (color version of figure is available online).
Fig 5: C-terminal 84aa fragment of CKB protein inhibits Vimentin promoter activity, focus formation and migration of PC3 cells. (A) (Top) GST-tagged AKT proteins were pull down by glutathione sepharose beads from 293T cells co-transfected with plasmids for GST-tagged AKT FL and Flag-tagged CKB-84aa (298aa-381aa), as indicated. Immunoblots for Flag and GST were shown. (Bottom) Immunoprecipitation assay using anti-Flag antibody from 293T cells co-transfected with plasmids for GFP-AKT-PH and Flag-tagged CKB C-terminal 84aa fragment. Immunoblots for Flag and GFP were shown. (B) Cell proliferation assay measured by alamar blue in 293T cells transfected with Flag empty vector, Flag-tagged CKB-185aa-381aa or Flag-tagged CKB-298aa-381aa (84aa) plasmids. (C) Luciferase activity in lysates co-transfected with Flag vector, Flag-tagged CKB-185-381aa or Flag-tagged CKB-84aa plasmids, together with Vimentin promoter firefly luciferase reporter and pGL4.74 renilla luciferase plasmids in 293T cells. Twenty-four hours later, cells were lysed for measuring luciferase activity. **P < 0.01, ***P < 0.001 from 2-sided t test comparing either CKB construct to vector control (triplicates). (D) Representative immunofluorescence images for GFP-AKT-PH (green), Flag-CKB 84aa fragment (red) and nuclei (blue). PC3 cells expressingGFP-AKT-PH were transfected with plasmid for Flag-CKB-84aa. White arrows indicate the PC3 cells transfected with Flag-CKB-84aa construct. Untransfected cells in the same wells serve as controls. Quantifications of GFP-AKT-PH signal ratios of membrane vs cytoplasm in untransfected (not red) and transfected (red) cells are presented in Figure S6B. (E) Focus formation assay of PC3 cells infected with lentivirus carrying either vector control or CKB-84aa. (F) Cell migration determined by Boyden chamber assay in PC3 cells infected with lentivirus carrying either vector control or CKB-84aa. Quantifications of focus formation and migrations (triplicates) are in Figure S6C-D. These experiments have been repeated at least twice, which has yielded same conclusions. Results from a representative experiment are shown (color version of figure is available online).
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