Fig 1: AKT inhibitor and CKB overexpression reverse EMT induced by CKB suppression. (A) RT-qPCR result of CKB expression in the indicated 4 cell lines treated with DMSO or 15uM AKT inhibitor MK-2206 for 24 h. Expression pattern for epithelial marker Occludin from these experiments is in Figure S4B. (B) Parental PC3 cells (wild type, WT), PC3 with CKB CRISPR-Cas9 knockout (CKB KO), or PC3 with CKB knockdown (shCKB-1) were treated with DMSO or 3uM of AKT inhibitor MK-2206 for 48h. Immunoblots for indicated proteins were shown. (C) qPCR analysis for indicated genes normalized to beta-Actin in PC3 parental cells (WT), CKB knockout cells (CKB KO), and CKB-knockout cells re-expressing CKB cDNA (CKB KO + CKB). *P < 0.05, **P < 0.01 comparing CKB KO to CKB KO + CKB. (D) Cell morphology were monitored by phase-contrast microscopy in PC3 WT, CKB KO and CKB KO + CKB cells. (E) Boyden chamber migration assay for PC3 WT cells, PC3 CKB KO cells and PC3 CKB KO + CKB cells. (F) Quantitative results of the migrations in E, based on triplicates. ***P < 0.001, ****P < 1 × 10-4, *****P < 1 × 10-5. (G) Alamar blue cell proliferation assay for PC3 WT, CKB KO and CKB KO + CKB cells cultured in media with reduced FBS (1.25%) at day 1, 3, 5 and 7 (quadruplicates). **P < 0.01, ***P < 0.001 comparing CKB KO cells, CKB KO + CKB cDNA cells with WT cells at corresponding time points. ^^P < 0.01 comparing CKB KO with CKB KO + CKB cells at day 7. These experiments have been repeated at least twice, which has yielded same conclusions. Results from a representative experiment are shown.
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: 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).
Fig 4: CKB overexpression inhibits EMT, migration and tumor growth of prostate cancer cells. (A) Luciferase activity in 293T cells 36 h after transfection with GFP (control, Ctrl) or CKB cDNA plasmid, together with a Vimentin promoter firefly luciferase (Fluc) reporter and a renilla luciferase (Rluc) control construct. Relative fold change of the Fluc/Rluc ratio was calculated and plotted as means ± SD. **P < 0.01 from triplicates. (B) Immunoblotting for indicated proteins in PC3 cells expressing GFP or CKB cDNA. (C) qPCR analysis of mRNA levels of indicated genes normalized to beta-actin in LN3 cells expressing GFP or CKB. (D) Anchorage-independent growth in a 24-well ultra-low attachment plate. (E) Cell migration as determined by Boyden chamber assay. Serum starved PC3-Ctrl and -CKB cells were treated or not treated with 20 ng/ml of EGF for 8 hr, followed by migration assays. Quantifications from duplicate experiments are in Supplementary Figure S3A. (F) 1 × 106 PC3-luciferase cells expressing GFP (Ctrl) or CKB cDNA were implanted subcutaneously in male NOD/SCID mice (n = 8 or 9 mice in either group). Xenograft tumor growth was monitored by bioluminescence imaging at day 1, day 42 and 70. Fold changes relative to day 1 were calculated and plotted as means ± SD. ***P < 0.001 from comparing the control and CKB groups at day 70.
Fig 5: 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).
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