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Figure 5 | Molecular Cancer

Figure 5

From: Genetic inactivation of the Fanconi anemia gene FANCC identified in the hepatocellular carcinoma cell line HuH-7 confers sensitivity towards DNA-interstrand crosslinking agents

Figure 5

Abrogation of the ICL-hypersensitivity phenotype in HuH-7 through exogenous FANCC expression. (A) Immunoblotting for the detection of FANCC-HA in parental HuH-7 cells (HuH-7) and HuH-7-derived clones retrovirally transduced with either empty vector-pMSCV (HuH-7/ev) or with pMSCV-FANCC (HuH-7/FANCC). HeLa cells transduced with pMSCV-FANCC were used as controls. CULLIN-1 served as loading control. FANCC-HA was detectable only in HuH-7/FANCC and control HeLa/FANCC cells. (B) FANCD2 monoubiquitination in parental HuH-7, HuH-7/ev and HuH-7/FANCC cells, respectively, either treated with MMC at 100 nM for 24 h or left untreated. RKO cells and derived RKO FANCC-/-/- cells were used as controls. β-ACTIN served as loading control. Monoubiquitinated FANCD2 was detectable only in HuH-7/FANCC and parental RKO control cells and was inducible upon MMC-treatment, confirming correction of proximal FA pathway function through exogenous FANCC expression in HuH-7. (C) FANCD2 nuclear focus formation in parental HuH-7, HuH-7/ev and HuH-7/FANCC cells either treated with IR at 15 Gy for 6 h or left untreated. FANCD2 focus formation was detectable only in HuH-7/FANCC cells. (D) Cell proliferation assays to compare the sensitivity of parental HuH-7, HuH-7/ev and HuH-7/FANCC cells towards ICL- and non-ICL-chemotherapeutic agents. HuH-7/FANCC cells were less sensitive towards treatment with ICL-agents than parental and HuH-7/ev control cells (upper panel: MMC, cisplatin, melphalan), with IC50 ratios ranging from 3.4- to 4.2-fold (arrows, indicated in red), whereas sensitivity towards non-ICL-agents (lower panel: 5-FU, doxorubicin, etoposide) remained virtually unchanged. Error bars represent SEM of three independent experiments.

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