Figure 3

Gene complementation studies identifying an inactivating FANCC nonsense mutation in HuH-7. (A) FANCD2 monoubiquitination in FA pathway-proficient control HeLa cells and HuH-7 cells transduced with the indicated FA or control cDNA constructs. All cultures were exposed to MMC at 150 nM for 15 h. RAD50 served as loading control. Only transduction with FANCC restored FANCD2 monoubiquitination in HuH-7 cells. (B) FANCC immunoblotting using the indicated cell lysates. The nuclear protein p84 served as loading control. Only HuH-7 cells transduced with FANCC and FA pathway-proficient Hep3B control cells displayed FANCC protein. (C) Cell cycle distributions of HuH-7 cells transduced with the indicated FA or control cDNA constructs. All cultures were exposed to MMC at 33 nM for 15 h. Only transduction with FANCC abrogated the pronounced G2 cell cycle arrest upon MMC in HuH-7 cells. (D) Chromatogram of the homo- or hemizygous FANCC mutation (arrow) identified in HuH-7 cells. (E) RT-PCR using three different primer pairs amplifying a region from FANCC non-coding exon 1 to coding exon 5, or from coding exons 3 to 13, or from coding exons 7 to 13, respectively, using cDNA from five HCC cell lines. Only HuH-7 cells exhibited aberrant mRNA splicing lacking exon 6. (F) Direct sequencing of the two distinct RT-PCR products confirmed that the product having the correct size represented the reference FANCC sequence except for the c.553C > T mutation, whereas the shorter product lacked exon 6, exhibiting a direct exon 5/7 junction.