P19arf

Cells were harvested and washed twice with PBS. Cell lysis was performed on ice for 25 min, in RIPA buffer (150 mM NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate, 50 mM Tris-HCl, pH 8.0) containing a protease inhibitory cocktail (Roche). Insoluble material was pelleted by centrifugation at 16,000× g at 4°C for 3 min. Protein concentrations were determined using the Bradford assay (Bio-Rad). Thirty micrograms extract was mixed with 4× Laemmli buffer (40% glycerol, 240 mM Tris/HCl, pH 6.8, 8% SDS, 0.04% bromophenol blue, 5% β-mercaptoethanol), denatured at 96ºC for 5 minutes, separated by SDS-PAGE, and transferred to nitrocellulose membranes (PROTRAN-Whatman, Schleicher&Schuell). The membranes were blocked with 5% non-fat dry milk in TBS-T for 60 min, incubated with primary antibodies overnight at 4°C, washed three times with TBS-T for 10 min, incubated with the peroxidase-conjugated secondary antibody (1:2000; Amersham Biosciences) in TBS-T with 5% non-fat dry milk for 60 min, and washed three times with TBST for 10 min. Immunoreactive proteins were detected using Supersignal West Dura HRP Detection kits (Pierce). The primary antibodies used were: p16^Ink4a (Santa Cruz); p19^Arf (Ab80 Abcam); p15^Ink4b (Santa Cruz); β-catenin (clone 14, BD Biosciences); p53 (sc-6243 Santa Cruz); p21 (BD Pharmigen); c-Myc (sc-764 Santa Cruz); β-actin (ab8226, abcam).

Cells were reprogrammed in 6-well plates on 18 mm circular coverslips (Fisher Scientific) for confocal imaging, or directly on the plastic for whole well imaging, both coated with gelatin. Cells were fixed with 4% Paraformaldehyde for 10 minutes, permeabilized in 0.1% Triton-X in PBS for 45 minutes, blocked in 5% normal goat serum in PBS for 1 hour at room temperature, and then stained in blocking solution with primary antibody overnight at 4°C. The next day, secondary antibody was applied in blocking solution for 1 hour at room temperature. Slides where then mounted with prolong gold with or without DAPI (Life technologies). The following antibodies were used with indicated dilution: p19ARF (1:300, Abcam, Ab80), E-CADHERIN-eFluor 660 (1:300, eBioscience, #50-3249-82), CD44-allophycocyanin (APC) conjugate (1:300, eBioscience, #17-0441), pSmad3 (1:100, Cell Signaling, C25A9). For confocal microscopy, all imaging was performed with a Leica TSC SP2 and processed using Adobe Photoshop. For whole well colony and cell counting, the sistched whole well images were taken and analyzed using the Celigo S Cell Cytometer (Nexcelom).

Cells were incubated in cell lysis buffer (no. 9803; Cell Signaling Technology, Danvers, MA) followed by centrifugation at 13,000 rpm for 10 minutes. Supernatants were collected, and protein concentrations were determined using the Bradford Assay (Bio-Rad, Hercules, CA). Protein lysates were electrophoresed on Tris-glycine SDS polyacrylamide gels and transferred onto Immobilon-P membrane. Incubation with antibodies was performed according to Cell Signaling Technology-recommended procedures and proteins were visualized using ECL (Pierce). p16^Ink4A, p19^ARF, p21, Total-p53, cdc2 were from Abcam. Phospho-p53(Ser15), Cyclin B1, Phospho-SAPK/JNK(Thr183/Tyr185), Total-SAPK/JNK and β-Actin were from Cell Signaling Technology.

Flag-Bmi1 (WT or K88R) and Flag-GFP-Bmi1 (WT or K88R) were generated using standard cloning procedures. K88R mutation was introduced into Bmi1 by site directed mutagenesis using QuikChange Site-Directed Mutagenesis Kit (Agilent California, USA). HA-SUMO1, HA-UBC9, RGS-SENP1 and RGS-SENP1m were previously described45 (link)48 (link). Anti-FLAG (mouse; #F1804) and anti-HA(mouse; #H3663) antibodies were from Sigma; anti-RGS-his (mouse; #34610) antibody from QIAGEN; anti-H-Ras (mouse; #sc-53959) and p16^Ink4a (mouse; #sc-1661) antibodies from Santa Cruz; anti-SUMO1 (rabbit; #ab5316), H3K27me3 (mouse; #ab6002) and p19^Arf (rabbit; #ab80) antibodies from Abcam; anti-Bmi1 (mouse; #05–637), γH2AX (mouse; #05–636) and uH2AK119 (mouse; #05–678) antibodies from Millipore.