Fig 2: Depletion of CHKA suppresses the invasive and metastatic potential of CRC cells in vitro and in vivo(A) Effects of CHKA knockdown on the migration and invasion of HCT116 or SW620 cells were determined by the transwell migration assay and matrigel invasion assay, respectively. Representative results are shown. (B) Plots for panel A are presented as mean ± SEM of data from three independent experiments. (C) Lung metastasis tumor model assays. 1 × 106 of HCT116 control or CHKA-depleted cells were injected into the tail vein of nude mice (n = 8). Ten weeks post inoculation, mice were sacrificed and metastatic tumor colonies in the lung were examined microscopically. Representative images of H&E staining of lung metastatic nodules in each group are shown. (Magnification, left panel, ×100; right panel, ×200) (D) The number of metastatic nodules in the lungs of each group is presented as mean ± SEM. (E) Kaplan-Meier curves for overall survival of mice in each group. The p-value was determined using the log-rank test. *p < 0.05.

Fig 3: PSMA-targeted CHKA inhibitor (CK147) concentration trend (72 h) in medium supplemented with 10% FCS (60 ± 0.3% intact parent at 72 h); n = 3, mean ± SD.

Fig 4: Mitochondria dynamic changes upon CHK inhibitionOxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of HCT116 cells following 24 h pre-treatment with ICL-CCIC-0019 (A), and following direct addition of ICL-CCIC-0019 (B). Vertical lines indicate the time of injection of ATP synthase inhibitor oligomycin (0.3 μg/mL), the pharmacological uncoupler FCCP (0.6 μM), complex III inhibitor antimycin (5 μg/mL), and complex I inhibitor rotenone (5 μg/mL) and ICL-CCIC-0019 (1 μM). (C) Effect of ICL-CCIC-0019 on AMPK activation in HCT116 cells; phosphorylated acetyl-coA carboxylase (pACC), AMPK phosphorylation at Thr172 (pT172), AMPK beta 1 and 2 subunits (B1 & B2) and citrate synthase (CS). (D) Effect of ICL-CCIC-0019 on citrate synthase in A459 WT and isogenic A549 Rho cells. (E) Effect of ICL-CCIC-0019 (5 μM, 24 h) on mitochondrial networks. Cells were stained with anti-mitochondrial antibody (green) and DAPI (blue). (F) Effect different inhibitors (10 μM each for 24 h) on mitochondria membrane potential measured by the tetramethylrhodamine ethyl ester dye method.

Fig 5: Increased expression levels of CHKA in CRC cell lines and clinical samples(A) Relative expression levels of CHKA mRNA in NCM460 and CRC-derived cell lines were determined by real-time qPCR methods. Gene expression results were normalized by internal control β-actin. (B) Protein expression levels of CHKA in NCM460 and CRC-derived cell lines were determined by western blot assay. β-actin was used as a loading control. (C) Correlations between relative expression levels of CHKA mRNA and protein in the cell lines examined. (D–E) Relative expression levels of CHKA mRNA in 63 paired human primary CRC tissues and adjacent nontumor tissues were determined by real-time qPCR. Gene expression results were normalized by β-actin. (T, tumor tissues; N, adjacent nontumor tissues) (F) CHKA protein expression in paired tumor and adjacent nontumor tissues was determined by western blot assay. (G) Representative immunohistochemical expression patterns of CHKA in cancerous and adjacent normal mucosa tissues are shown. (Magnification, upper panel, ×100; lower panel, ×400) (H) Comparison of IRS values of CHKA expression in 234 paired primary CRC tissues and adjacent nontumor tissues. (I) Percentage of cases with different staining intensity of CHKA in the tumor or adjacent nontumor tissues in the study cohort. (J) Comparison of the expression levels of CHKA mRNA in the 63 fresh-frozen CRC tissue samples from patients with different clinical stages (early stage, n = 35; advanced stage, n = 28). (K) Comparison of the IRS values of CHKA expression in the 234 CRC tissue samples from patients with different clinical stages (early stage, n = 146; advanced stage, n = 88). *p < 0.05; **p < 0.01.