Fancd2

Cells were washed in phosphate‐buffered saline (PBS) and lysed in lysis buffer (50 mm Tris/Cl, pH 7.4, 150 mm NaCl, 0.3% Igepal CA‐630, 0.2% Triton X‐100, 10 mm NaF, 1 mm sodium orthovanadate, and protease inhibitors). Lysates were cleared by centrifugation and supernatants were obtained. Equal amounts of lysate were resolved on 3–8% NuPAGE Tris‐acetate gel (Invitrogen, Carlsbad, CA, USA). Resolved proteins were electro‐transferred onto nitrocellulose membrane (GE Healthcare Europe, Freiburg, Germany) and detected by immunoblotting with antibodies to FANCD2 (NB100‐182; Novus Biologicals, Littleton, CO, USA), phopho‐AMPKα (#2535, Cell Signaling Technology, Danvers, MA, USA), AMPKα (#2532, Cell Signaling Technology), FANCA (A301‐980A, Bethyl Laboratories, Montgomery, TX, USA), p53 (OP43, Calbiochem), p21^Cip1 (#2947, Cell Signaling Technology), PARP‐1 (#9542, Cell Signaling Technology), and p15^Ink4B (#4822, Cell Signaling Technology). Protein bands were visualized with the ECL Prime kit (GE Healthcare UK, Little Chalfont, UK).

Cells were cultured on 12 mm diameter microscope cover glasses, permeabilized with PBS containing 0.1% Triton X-100 for 3 min, and then fixed with 3.7% formaldehyde (Sigma) for 10 min at room temperature. Cells washed by PBS were extracted with 0.5% NP-40 (USBiological, Salem, MA, USA) for 5 min at room temperature. After they were washed, cells were blocked with blocking buffer (0.2% gelatin and 0.5% BSA in PBS) for 1~2 h at room temperature and then incubated with primary antibodies, such as FANCA (Merck Millipore: MABC557), FANCD2 (Novus Biologicals: NB100-182), PML (Santa Cruz Biotechnology: sc-966), or γH2AX (Cell Signaling Technology: #2595) overnight at 4 °C. Samples were washed three times for 10 min with blocking buffer. Secondary antibodies (Abcam, Cambridge, UK) mouse 488 (Abcam: ab150109), rabbit 594 (Invitrogen: A21207), mouse 594 (Abcam: ab150112), and rabbit 488 (Abcam: ab150061) were diluted 1:2500 and incubated for 1 h at room temperature in the dark. Finally, cells were washed three times for 30 min and mounted in Vectashield^® containing DAPI (Vector Laboratories, Burlingame, CA, USA).

Cells were grown on no. 1 glass coverslips and fixed with 4% methanol-free paraformaldehyde (PFA) in PBS. Cells were then permeabilized in PBS plus 0.1% Triton X-100 (PBT) and blocked with normal goat serum plus 0.15 Triton X-100 (NGS-T) followed by incubation with primary antibody in NGS-T overnight at 4°C in a humidity chamber. Coverslips were washed in PBS and incubated with Alexa Fluor secondary antibody (Invitrogen) for 30 min at 37°C. After PBS washes (3×), cells were counterstained with DAPI (4′,6-diamidino-2-phenylindole) and mounted with Gelvatol. The antibodies used were FANCD2 (Novus Biologicals, Inc., catalog no. NB100-182), BRCA1 (Oncogene catalog no. OP92), γH2AX-Ser139 (Millipore catalog no. 05-636), and p-SMC1-Ser957 (Cell Signaling catalog no. 4805). For 4-color immunofluorescence, following secondary washes, cells were incubated with Alexa Fluor 647-γH2AX-pS139 (BD Pharmingen no. 560447) in a light-protected humidity chamber for 1 h at room temperature. Coverslips were washed in PBS, counterstained with DAPI, and mounted in Gelvatol. Cells were imaged using a Zeiss Axioscope and analyzed using ImageJ.