(a) Effect of restoration of PRTFDC1 on growth of oral squamous-cell carcinoma (OSCC) cells. A Myc-tagged construct containing PRTFDC1 (pCMV-3Tag4A-PRTFDC1) or empty vector (pCMV-3Tag4A-mock) as a control was transfected into NA or OM-2 cells, which lack expression of the PRTFDC1 gene. Upper: western-blot analysis using 5 μg of protein extract from cells 48 h after transfection and anti-Myc antibody demonstrated that transiently pCMV-3Tag4A-PRTFDC1-transfected cells expressed Myc-tagged PRTFDC1 protein. Middle: 3 weeks after transfection and subsequent selection of drug-resistant colonies in 6-well plates or 100-mm dishes, the colonies formed by PRTFDC1-transfected cells were less numerous than those formed by mock-transfected cells. Lower: quantitative analysis of colony formation. Colonies larger than 2 mm were counted, and results are presented as the means±s.d. (bars) of three separate experiments, each performed in triplicate. Statistical analysis used the Mann–Whitney U-test: *P<0.05 versus empty-vector transfected cells. (b) Effects of knockdown of endogenous PRTFDC1 on growth of OSCC cells. Upper: Quantitative real-time RT–PCR analysis of PRTFDC1 mRNA after transfection of 50 nM of PRTFDC1-specific small interfering RNA (siRNA) (PRTFDC1-siRNA) or a control siRNA for the luciferase gene (Luc-siRNA) into OSCC cell lines expressing PRTFDC1 (HO-1-N-1 and HSC-4). Relative expression levels of PRTFDC1 mRNA in PRTFDC1-siRNA-treated cells normalized with those in Luc-siRNA-transfected counterparts at indicated times were calculated. Middle: The numbers of viable cells after transfection of 50 nM of each siRNA were assessed at the indicated times by water-soluble tetrazolium salt assay. The data presented are the means±s.d. of triplicate experiments. Statistical analysis used the Mann–Whitney U-test: *P<0.05. Lower: The population in each phase of cell cycle was assessed by fluorescence-activated cell sorting using HO-1-N-1 cell line 72 h after transfection of PRTFDC1-siRNA or Luc-siRNA. The data presented are the means±s.d. of triplicate experiments. Statistical analysis used the Mann–Whitney U-test: *P<0.05.