Immunofluorescence-staining

To analyze RAD51 and γH2AX foci formation in reprogamming cells, CCD-1079sk cells were reprogramed with Yamanaka 4 factors or combined with other factors as described in the section of Lentiviral transduction and reprogramming culture. At 9 and 12 dpi, cells were collected and washed with hES culture medium and then plated on a Coverglass For Growth (Fisher Scientific, FIS12-545-82) which were covered with gelatin. After 6 h of culture, cells were rinsed with PBS and incubated with Permeate Buffer (0.2% Triton X-100 in PBS) at room temperature for 3 min. Cells on coverslips were rinsed twice with ice-cold PBS and then fixed with 4% PFA (Sigma) on ice for 15 mins. Cells were washed twice with PBS, and permeabilized with PBST (0.2% triton X-100 in PBS) at room temperature (RT) for 15 mins. After blocking in FDB (0.2% Triton X-100, 2% donkey serum, 3% bovine serum albumin, 1 × PBS) for 30 mins at RT, the coverslips were incubated with primary antibody for 1 hour (h) at RT, followed by 3 × 3 mins washes with PBST. A secondary antibody (Invitrogen) (1:400 diluted in blocking solution) was added and incubated for a further 1 h at RT. After a total 3 rounds of washing with PBST quickly, the coverslips were mounted on slides with a mount medium containing DAPI (VectaShield). RAD51 polyclonal antibody (ct-1201, Cell Application) and γH2AX S139 monoclonal antibody (#05-636, Millpore) were used for immunostaining. Total number of γ-H2AX/Rad51 positive cells were counted from randomly picked up 150 cells in each sample.

iPS colony immunostaining was performed as described above. The antibodies were used as follow: Anti-SOX2 (rabbit IgG, 1:1000, Millipore, AB5603), Anti-SSEA4 (mouse IgG, 1:400, DSHB, MC-831-70), Anti-Nanog (goat IgG, 1:150, R&D, AF1997), Anti-Tra-1-60 (mouse IgM, 1:150, Millipore, MAB4360), Anti-Tra-1-81 (mouse IgM, 1:150, Millipore, MAB4381), Anti-mouse IgM (546 nm, goat IgG, 1:1000, Invitrogen, A21045), Anti-goat IgG (594 nm, donkey IgG, 1:1000, Invitrogen, A11058), Anti-mouse IgG (647 nm, donkey IgG, 1:1000, Invitrogen, A31571), Anti-rabbit IgG (647 nm, donkey IgG, 1:1000, Invitrogen, {"type":"entrez-protein","attrs":{"text":"A31573","term_id":"87384","term_text":"pir||A31573"}}A31573).