Pcmv7 3xflag zeo vector

siRNA-resistant wild-type human Rev3, or catalytic mutant (D2781A D2783A) Rev3 cDNAs were cloned into the pCMV7-3xFlag-zeo vector (Sigma-Aldrich). The vectors were each transfected into SV40 transformed GM637 HFs by lipofectamine 2000 reagent (Invitrogen). After 24 h incubation, 0.5 μg of zeocin (GenDEPOT) was added to the culture media. After an additional 3 d of incubation, the cells were washed with PBS buffer and were continuously cultured in media containing 250 ng of zeocin for ∼2 wk. Protein expression was verified by Western blot analysis (Fig S1D).

Full-length human Polη or Polλ ORFs were cloned into the pCMV7-3xFlag-zeo vector (Sigma-Aldrich) and stably expressed into XPV HFs or GM637 HFs, respectively. For Rev7 foci formation, Rev7 antibody (BD Biosciences) was used for immunofluorescence in GM637 HFs. Cells were suspended and treated with siRNA and cultured on a coverslip in six-well plate with 50% confluence. After 48 h, cells were treated with UVC (30 J/m²). For UV irradiation, cells were washed with PBS buffer and irradiated with UVC light in the presence of PBS buffer. After irradiation, fresh growth medium was added to cells and the cells were incubated for 6 h. After washing with PBS buffer, cells were fixed with 4% paraformaldehyde for 30 min. Fixed cells were permeabilized with 0.2% Triton x-100 in PBS buffer. Primary FLAG antibody (Sigma-Aldrich) or Rev7 antibody (BD Biosciences) were added to cells in PBST (0.1% Tween 20 in PBS) containing 3% BSA. Nuclear staining was performed with DAPI (Molecular Probe) in PBS buffer for 20 min. The fluorescent images were visualized and captured by fluorescence microscopy (Nikon fluorescence microscope).