Fig 1: MtCK1 regulates human osteoclast mitochondrial bioenergetics and bone resorption A hMDMs were transfected with either nontargeting siCTRL or siRNA targeting CKMT1, differentiated into osteoclasts, and cell lysates collected for MtCK1, CKB, ZEB1, c-SRC, and CTSK immunoblotting (n = 3).B Mitochondrial creatine kinase activity of siCTRL- or siCKMT1-transfected osteoclasts cultured on plastic (n = 3).C Phosphocreatine/creatine (PCr/Cr) ratio of siCTRL- or siCKMT1-transfected osteoclasts cultured on plastic as determined by LC/MS analysis (n = 3).D, E ATP levels (D) and RhoA activity (E) of siCTRL- or siCKMT1-transfected osteoclasts.F Mitochondrial function parameters analyzed by XF Cell Mito Stress Assay in siCTRL- or siCKMT1-transfected osteoclasts after sequential treatment of compounds modulating mitochondrial function (n = 4).G, H siCTRL- or siCKMT1-transfected human osteoclasts were cultured atop bone slices for 3 days, stained with phalloidin (red). Osteoclasts were removed and resorption pits visualized by WGA-DAB staining (G). Scale bar, upper 20 µm, middle and lower 100 µm. The actin ring area per cell and resorption pit area were quantified (H; n = 6).I TRAP, resorption pits visualized with WGA-DAB staining, and phalloidin staining in wild-type osteoclasts treated with vehicle, and Zeb1 ?M/?M osteoclasts treated with vehicle or 200 µM cyclocreatine cultured on bone slices with the number of TRAP+ MNCs, resorption pit area, and actin ring area per cell quantified (n = 6). Data information: Bars and error bars represent mean ± SEM. Data are representative of at least three independent experiments with biological replicates. Data were analyzed using one-way ANOVA with Bonferroni correction (B–F, H, I). ns, not significant; *P < 0.05, **P < 0.01. Source data are available online for this figure.
Fig 2: 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 3: 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 4: 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 5: CKB interacts with AKT PH domain through its C-terminal. (A) Schematic of GST-tagged AKT full-length (FL) and truncation mutants (left). The 293T cells were transfected with cDNA vectors for GST-tagged AKT FL or truncations, together cDNA plasmid for Flag-tagged CKB FL protein. GST-tagged AKT proteins were immunoprecipitated by glutathione sepharose beads from co-transfected cells, followed by immunoblotting for Flag and GST (right). (B) PIP3 coated agarose beads were incubated with purified His-tagged AKT (2ug) and/or His-tagged CKB (0, 1 or 2ug) as indicated. PIP3 binding proteins were pulled down. Immunoblots for AKT and CKB were shown. (C) Schematic of Flag-tagged CKB FL and truncation mutants (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 FL or truncations as indicated (middle). Immunoblots on input whole cell lysates are in the bottom. (D) 293T cells were co-transfected with GST-tagged AKT vector and indicated amounts of Flag-tagged CKB truncation vectors. GST-tagged AKT proteins were pull down by glutathione sepharose beads from these cells. Immunoblots for Rictor, mTOR, Flag and AKT were analyzed. (E) Representative immunofluorescence images for GFP-AKT-PH fusion protein (green), Flag-CKB FL protein or Flag-CKB truncations (red) and nuclei (blue). PC3 cells expressing GFP-AKT-PH were transfected with Flag-CKB plasmids. White arrows indicate the PC3 cells transfected with the corresponding CKB full length or truncated cDNA constructs. Untransfected cells in the same wells serve as controls. Additional representative images are in Supplementary Figure S5. Quantifications of GFP-AKT-PH signal ratios on membrane vs cytoplasm in multiple untransfected and transfected cells are presented in Figure S6A. These immunoblotting and IF experiments have been repeated twice, which has yielded same conclusions. Results from a representative experiment are shown (color version of figure is available online).
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