Ab108928

The separated tumor tissues were fixed in formalin, dehydrated in ascending grades of ethanol, embedded in paraffin, and sectioned at 4 μm thickness. After dewaxing and dehydration, the tissue sections were incubated in 3% hydrogen peroxide for 10 min and microwaved for antigen removal for 15 min. After blocked with bovine serum albumin, the sections were incubated with FANCD2 antibody (ab108928, 1:1000, Abcam) overnight at 4°C, followed by secondary antibody (1:500) at 37°C for another 30 min. Then sections were stained by diaminobenzidine and counterstained by hematoxylin. Finally, all tissue sections were observed under an inverted fluorescence microscope (Olympus, Tokyo, Japan).

Slices were dewaxed and hydrated. Endogenous catalase was removed by H₂O₂. The slices were blocked for 30 min at 37°C with 5% BSA solution (P0220, Beyotime). Following that, the sections were reacted with primary antibody [eIF5A (1 : 250, ab32443, Abcam); FANCD2 (1 : 100, ab108928, Abcam); SLC7A11 (1 : 500, ab216876, Abcam); HSPB1 (1 : 500, ab109376, Abcam)] overnight at 4°C. The sections were incubated with the secondary antibody for 30 min. Then the slides were treated for 5 min with diaminobenzidine (DAB, Beyotime, China). After restaining for 5 min with hematoxylin, the slices were dehydrated, made transparent, and finally sealed with neutral resin. The results were observed with an optical microscope (Olympus Corporation).

IF staining was performed as previously described8 (link). DSB foci were detected by immunostaining with a monoclonal antibody to γH2AX (EMD Millipore; 05-636, 1:400), a polyclonal antibody to 53BP1 (Novus Biologicals; NB100-904, 1:100) or FANCD2 (Abcam; ab108928, 1:50). Localization studies were performed using antibody to PTEN (Cell Signalling; 9188, 1:100) or CK2β (Santa Cruz; sc-12739, 1:50) followed by incubation with a polyclonal goat anti-mouse or rabbit IgG Alexa Fluor 488/568 (Invitrogen) at 1:400 for 1 h. Cover glasses were mounted in Vectashield mountant with DAPI (Vector Laboratories) as nuclear stain. Images were captured using oil immersion 63 × objectives Zeiss Axio Imager A1/Axio Cam MRC and Axiovision LE software (Carl Zeiss, Oberkochen, Germany).

Cells were solubilized in RIPA lysis buffer (P0013B, Beyotime Biotechnology, Shanghai, China), and the supernatant was collected after centrifugation at 12,000 rpm for 20 min. Total protein concentration was detected by BCA assay (P0009, Beyotime Biotechnology, Shanghai, China). Proteins were then separated using 12% SDS-PAGE (P0012A, Beyotime Biotechnology, Shanghai, China), and transferred onto polyvinylidene fluoride membranes (PVDF, 88585, ThermoFisher Scientific, California, USA). Upon completion of blocking step in 5% skimmed milk for 1 h, the membranes were incubated overnight at 4 °C with primary antibodies Fancd2 (1:1000, ab108928; Abcam, Cambridge, UK), solute farrier family 7 member 11 (SLC7A11, 1:1000, ab175186; Abcam, Cambridge, UK), and glutathione peroxidase 4 (GPX4, 1:1000, ab252833; Abcam, Cambridge, UK). On the next day, the membranes were incubated with HRP-conjugated secondary antibodies (1:5000, ab205719; Abcam, Cambridge, UK) for 2 h. After that, the protein bands were visualized using the ECL Western blotting Kit (32109, ThermoFisher Scientific, California, USA). Finally, the relative expression of proteins was calculated using GAPDH as an internal reference.

Nuclear FANCD2 foci were measured in high throughput by quantitating DAPI stained cell nuclei of either SUM149 or SUM149 B1.S* cell lines using automated confocal imaging (CX7 LZR, ThermoFisher Scientific). Control DMSO, treated cells reached 85% confluence at the conclusion of 2 days of 1 µM drug exposure. FANCD2 foci were detected with immunofluorescence using rabbit anti-human FANCD2 (abcam, ab108928). 1 µM was selected in order to have sufficient nuclei for FANCD2 foci quantification, as 10 µM compounds was too toxic for certain compounds.

Protein extraction was performed by washing the cell pellet with ice-cold PBS. Next, 1 mL of ice-cold RIPA lysis buffer (Sigma) and a protease inhibitor cocktail (Thermo Scientific, Rockford, IL, USA) were added and incubated on ice for 30 min. Then, 30 µg of cell lysates were resolved on 4–20% ExpressPluS PAGE Gel (GenScript, Piscataway, NJ, USA) following concentration measurements. The eBlot Protein Transfer device (GenScript, Piscataway, NJ, USA) was used to transfer the proteins onto a PDVDF Transfer Membrane (Thermo Scientific, Rockford, IL, USA). The membranes were washed and incubated for 1 h with a secondary anti-mouse antibody conjugated with HRP (Cell Signaling Technology, Danvers, MA, USA). Pierce ECL Western blotting Substrate (Thermo Scientific, Rockford, IL, USA) and BioRad Universal Hood II with a Chemiluminescence System (BioRad, Hercules, CA, USA) were used to visualize the result. Antibodies: Recombinant Anti-FANCD2 antibody [EPR2302] (abcam—ab108928) 1/1000; Recombinant Anti-Rad51D antibody [EPR16205] (abcam—ab202063) 1/1000; Recombinant Anti-β Actin antibody [EPR21241] (abcam—ab213262) 1 µg/mL; Recombinant Anti-Rad51D antibody [EPR16205] (ab202063) 1/1000.