Sc 20022

HeLa (originally from ATCC) and PD20 (kindly provided by the FARF repository) cells were grown in DMEM (D5796, Sigma) supplemented with 2.5–10% fetal bovine serum. Antibodies used were as follows: anti-FANCD2, 1:100 dilution (sc-20022, Santa Cruz Biotechnology); anti-FANCI, 1:1,000 dilution (FARF); anti-FANCA, 1:1,000 dilution (FARF); anti-Lamin B, 1:1,000 dilution (sc-6216, Santa Cruz Biotechnology); anti-Flag, 1:1,000 dilution (M5, F4042, Sigma-Aldrich); anti-Ubiquitin, 1:400 dilution (FK2,BML-PW8810, Enzo Life Sciences); anti-UHRF1, 1:1,000 dilution (sc-373750, Santa Cruz Biotechnology); and anti-a-Tubulin, 1:2,000 dilution (5829, Millipore).

Cells were collected and disrupted in lysis buffer (50 mM Tris pH 7.5, 20 mM NaCl, 1 mM MgCl₂, 0.1% SDS supplemented with protease and phosphatase inhibitors (Roche) and 0.1% endonuclease benzonase (Millipore)). After 10 min of incubation at room temperature, the lysates were combined with 4× Laemmli buffer containing β-2-mercaptoethanol and denatured by boiling. The proteins were separated by SDS-PAGE. The antibodies used were directed against FANCD2 (sc-20022, Santa-Cruz), actin (sc-1616, Santa-Cruz), vinculin (ab18058, Abcam), RAP80 (ab124763, Abcam), GAPDH (ab9484, Abcam), lamin B1 (ab20396, Abcam), histone H3 (ab1791, Abcam), Tip60 (sc-5725, Santa-Cruz), 53BP1 (MAB3802, Millipore), H4K16Ac (07-329, Millipore), H3K9Me3 (6F12-H4, Millipore), H3K9Ac (39585, Active Motif), H4K20Me2 (39174, Active Motif) and HA (26183, Thermo).

For immunofluorescence (IF) analyses, cells were seeded in four-well tissue culture slides (BD Biosciences) or on cover slips (Corning) and treated with MMC for 18 h. Cells were fixed in 4% w/v paraformaldehyde in PBS for 15 min on ice, followed by permeabilization for 5 min in 0.3% v/v Triton X-100 in PBS. Fixed cells were incubated with primary antibodies in 5% v/v goat serum, 0.1% v/v NP40, in PBS for 1 h, washed three times with PBS and then incubated with Alexafluor 488-conjugated anti-mouse or anti-rabbit secondary antibodies (Invitrogen) for 45 min. Cells were then counterstained and mounted in vectashield plus 406-diamidine-2-phenylindole dihydrochloride (DAPI) (Vector Laboratories) and visualized using a Zeiss AxioImager.A1 upright epifluorescence microscope with AxioVision LE 4.6 image acquisition software. Primary antibodies used for IF were anti-53BP1 (H300; Santa Cruz Biotechnology), anti-DNA-PKcs pS2056 (ab18192; Abcam), anti-FANCA (ABP6201; Cascade), anti-FANCD2 (NB100–182; Novus Biologicals and sc-20022; Santa Cruz Biotechnology), anti-FANCI (A300-212A; Bethyl Laboratories), anti-FANCM (Meetei and Deans CE56.1 antibodies), anti-FK2 (sc-8017; Santa Cruz Biotechnology), and anti-γH2AX (05–636; Millipore).

DNA damage induction was determined as described previously [26 (link)]. Briefly, cells were plated at an appropriate density and allowed to settle overnight. Media was supplemented with 1 at the dose stated and cells were allowed to grow for 4 days with constant exposure. Cells were trypsinised, collected and lysed in Cell Panel Lysis Buffer with complete protease and phosphotase inhibitors. Proteins were separated by gel electrophoresis and transferred to nitrocellulose membrane by Western blot. Membranes were probed, at a concentration of 1:1000 unless stated otherwise, for γH2AX (#2577, Cell Signaling Technology; 1:500), GAPDH (#3683, Cell Signaling Technology; 1:5000), phospho-ATM (Ser1981) (ab81292, Abcam) and FANCD2 (sc-20022, Santa Cruz Biotechnology).

The immunohistochemistry performed FANCD2 protein expression in normal and PAAD tissues. We detected HNRNPC expression using “tissue” and “pathology” of modules in HPA database. All results were confirmed by two pathologists independently. Regents as following: FANCD2: Santa Cruz Biotechnology Cat#sc-20022, RRID:AB_2278211, dilution 1:10.