Fig 1: Effect of creatine shuttle inhibition by DNFB on growth and stemness of CRC cells.(A) Effect of DNFB on cell growth. (B) Effect of pCr on DNFB-treated CRC cells. (C) Effect of DNFB (10 µM) on expression of stemness-associated genes detected by quantitative RT-PCR. ß-Actin was used as a loading control. (D) Effect of DNFB (10 µM) on sphere formation. Scale bar, 50 µm. (E, F) Effect of knockdown of CKB or MTCK on cell proliferation (E) and sphere formation (F). Error bars: standard deviation of three independent trials. Statistical significance was calculated using a two-tailed ordinary analysis of variance. Abbreviations: CKB: creatine kinase B; MTCK: mitochondrial creatine kinase; DNFB: dinitrofluorobenzene; CRC: colorectal cancer; OD: optimal density; pCr: phosphocreatine; C: untreated control.
Fig 2: Immunohistochemistry of CKB and MTCK in CRCs.(A, B) Immunohistochemical examination of CKB (A) and MTCK (B). Immunohistochemical activity is classified into grade 0–3. Grade 0 is equivalent to the expression in the normal colonic epithelium. (C) Histogram of expression grade. (D) Immunohistochemical examination of CKB and MTCK in case of well-differentiated adenocarcinoma (tub1), signet ring cell carcinoma (sig), primary tumor and liver metastasis of tub1 case. Scale bar, 100 μm. Abbreviations: CKB: creatine kinase B; MTCK: mitochondrial creatine kinase; CRC: colorectal cancer.
Fig 3: Creatine shuttle provides ATP for EGFR phosphorylation in EGF-treated HT29 cells.(A) Effect of DNFB (5 µM) and knockdown of MTCK or CKB on EGFR phosphorylation. Lower panels, effect of siMTCK and siCKB on expression of MTCK and CKB, respectively. (B) Semi-quantification of EGFR phosphorylation levels. (C) Effect of ATP on suppressed EGFR phosphorylation by DNFB (5 µM). (D) Effect of oligomycin on EGFR phosphorylation. (E) Effect of CyCr on creatine phosphorylation. (F) Effect of CyCr on EGFR phosphorylation. (G) Co-immunoprecipitation using anti-CKB antibody or anti-EGFR antibody to examine binding of CKB and EGFR. (H) Duolink® proximity ligation assay. Red, proximity signal of EGFR and CKB. Blue, DAPI. Scale bar, 10 µm. Error bars: standard deviation of three independent trials. Statistical significance was calculated using a two-tailed ordinary analysis of variance. Abbreviations: DNFB: dinitrofluorobenzene; EGFR: epithelial growth factor receptor; pEGFR: phosphorylated EGFR; EGF: epithelial growth factor; CKB: creatine kinase B; MTCK: mitochondrial creatine kinase; siC: short interfering RNA (siRNA) used as control; siMTCK: siRNA for MTCK; siCKB: siRNA for CKB; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; OM: oligomycin; CyCr: cyclocreatine; pCr: phosphocreatine; IP: immunoprecipitation; CB: Coomassie blue; WCL: whole cell lysate; DAPI: 4',6-diamidino-2-phenylindole.
Fig 4: Creatine kinase inhibitory effect of DNFB.(A) Protein fractionation of mitochondria (Mitochondrial) and mitochondria-free cytosol (Cytosol). MTCK and CKB were detected by western blotting. TOM20 and GAPDH were subjected as a mitochondrial marker and a cytosol marker, respectively. (B) Inhibition of creatine kinase activity (at 20 min) of CKB (cytosol fraction) and MTCK (mitochondrial fraction) in a DNFB concentration-dependent manner. (C, D) Inhibition of creatine kinase activity of CKB (C) and MTCK (D) in a time-dependent manner. Error bar, standard deviation of three independent trials. Statistical significance was calculated using a two-tailed ordinary analysis of variance. Abbreviations: CKB: creatine kinase B; MTCK: mitochondrial creatine kinase; DNFB: dinitrofluorobenzene; TOM20: translocase of the outer membrane 20; GAPDH: glyceraldehyde 3-phosphate dehydrogenase.
Supplier Page from Abcam for Anti-Creatine kinase B type antibody