Fig 1: CDC7-binding region is critical for the activation of the RAD54L promoter. (A) Top 10 genes correlated to CDC7 mRNA expression in TCGA dataset. Pearson correlation analysis was performed. (B) qRT-PCR analysis for CDC7 and RAD54L mRNA expression in U87 cells transduced with shRNA against CDC7 (shCDC7) or non-targeting control (shNT). (P < .05, n = 3 with t test). (C) Western blotting analysis of U87 cells transduced with shRNA against CDC7 (shCDC7) or non-targeting control (shNT). β-actin served as a control. (D) Flow cytometry analysis for apoptosis with Annexin V antibody and Propidium Iodide using U87 cells transduced with shNT or shCDC7. E. RT-PCR analysis for CDC7 and BUB1B mRNA expression in U87 cells transduced with shNT or shCDC7 with or without radiation treatment (12 Gy) (P < .01, with t tests). F. Luciferase activity assay showed shRNA-mediated-knockdown of CDC7 resulted in a marked decrease in transcription activity of RAD54L promoter in U87 cells(P < .01, with t tests).
Fig 2: CDC7 was functionally required for GBM proliferation both in vitro and in vivoA. qRT-PCR analysis of U87 cells transduced with shRNA against CDC7 (shCDC7) or non- targeting control (shNT) (P < .05, n = 3, with t test). B. Western blotting analysis of U87 cells transduced with shRNA against CDC7 (shCDC7) or non-targeting control (shNT). β-actin served as a control. C. In vitro cell growth assay showed shRNA against CDC7 (shCDC7) inhibited cell proliferation of U87 cells (P < .05, n = 6, with t test). D. Representative images of H&E stained mouse brain section after the intracranial transplantation of U87 cells transduced with shRNA against CDC7 (shCDC7) or non-targeting control (shNT). E. Kaplan-Meier analysis of nude mice harboring intracranial tumor derived from U87 cells transduced with shNT (n = 5), shCDC7 (n = 5) (P = .0066, with log-rank test).
Fig 3: CDC7 expression was highly enriched in GBM cells(A) Genome-wide transcriptome microarray analysis from Mao’s dataset shows that CDC7 is one of the most up-regulated kinase encoding genes in GBM samples compared to normal tissue. (B) Analysis of Mao’s dataset indicated that CDC7 was highly expressed in both mesenchymal and proneural subtypes of GBM compared with normal astrocytes. (C-D) Analysis of TCGA database indicated that CDC7 was highly expressed in all the 4 subgroups of GBM (classical, mesenchymal, neural and proneural) compared with non-tumor tissue. (E) qRT-PCR analysis showed BUB1B mRNA expression was elevated in 6 GBM cell lines (SHG44, T98G, U87, U373, U251 and U138) compared with normal astrocytes (NHA). (F) Western blotting analysis indicated that CDC7 protein expression was enriched in GBM tissue compared with normal brain from the paired patient samples. β-actin served as a control.
Fig 4: CDC7 expression was associated with poor prognosis in GBM patients. (A) Representative images of IHC stained patient samples. (B) Analysis of patient samples indicated the inverted correlation between CDC7expression and post-surgical survival of GBM patients.
Supplier Page from OriGene Technologies for CDC7 Human shRNA Lentiviral Particle (Locus ID 8317)