Fig. 4

PDHK1-241aa, but not circPDHK1, promotes the proliferation, migration and invasion of ccRCC cells in vitro and in vivo. A Colony formation assays, B CCK-8 assays and (C, D) EdU assays were performed to detect the cell viability and proliferation activity of Caki-1 and 786-O cells transfected with pLC5-ciR, circPDHK1 and circPDHK1 ATG mut vectors. Bars = 200 μm. E–G Transwell assay to detect cell migration and invasion of Caki-1 and 786-O cells transfected with pLC5-ciR, circPDHK1 and circPDHK1 ATG mut vectors. Bars = 100 μm. H, I wound healing assays were conducted to evaluate cell migratory abilities in Caki-1 and 786-O cells tansfected with pLC5-ciR, circPDHK1 and circPDHK1 ATG mut vectors. Bars = 100 μm. J Xenograft 786-O tumors in NSG mice were injected with circPDHK1 and circPDHK1 ATG mut overexpression lentiviruses. Tumor volumes were calculated every 2 d. At Day 18 after treatment, all mice were sacrificed, and (K) subcutaneous tumors were dissected and recorded. L Subcutaneous tumor was also photographed. (n = 5, each group). M Representative photographs of Ki-67 IHC staining in subcutaneous tumors. N We also established a lung metastasis model by injecting 6 × 10⁶ Caki-1 cells into the tail vein to evaluate the ability of cells to metastasize in vivo. Representative in vivo imaging of mice, photographs of whole lung tissues and hematoxylin–eosin (H&E) staining of lung metastatic nodules. (n = 3, each group) Bars = 200 μm. *P < 0.05; **P < 0.01; ***P < 0.001; ns, no significance