Supplementary figure A: Consort diagram.

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Supplementary material on anti-RAD51 antibody validation

Cell LinesA-549 and CALU-1 cells were grown in RPMI 1640 media (R5886 fa Sigma), with 10% FBS(A15-151 fa PAA), 1% sodium pyruvate 100mM (S8636 fa Sigma), 1% Penicillin-Streptomycin(K952 fa Amresco) and 1% L-Glutamine 200mM (25030 fa GIBCO). Cell lines were maintainedat 37°C in a humidified atmosphere at 5% CO2. After reaching the confluence of 60-70%, cellswere irradiated (4 Gy), and incubated at 37°C in a humidified atmosphere at 5% CO2 from 2 to 5hours. After incubation, cells were harvested and paraffin embedded cell blocks were prepared.

Cell block preparationCell suspension was centrifuged at 1200 rpm for 5 min. at room temperature and the cell-freesupernatant discarded. Cell pellet was resuspended in 50 ml medium and centrifuged at 1200rpm for 5 min. The cell pellet was again resuspended in 10 ml PBS, transferred in 15 ml falconvial and centrifuged at 1200 rmp for 5 min. The supernatant was removed. The cell pellet wasresuspended using the mixture of 450 μl citrate-plasma and 11,3 μl 1M calcium chloride andtransferred into 2ml tube. Clot was rapidly formed by adding of 45 μl of thrombin (120 NIHU/ml). The clot was transferred into a filter paper in histosette (for biopsy). After formalinfixation, clots were processed by paraffin embedding and haematoxylin-eosin (H&E) staining ofwhole sections.

Immunohistochemical staining of pre-extracted cell block sectionsCell blocks were cut for 4 mikrometer thick sections, deparaffinized in xylene and rehydratedwith serial dilutions of alcohol, after rehydration step sections were covered with cold proteinextraction buffer: 50mM Tris-HCL (pH7,5 ), 150mM NaCl, 1mM MgCl2 , 5% glycerol, 1mMDTT, 1% Triton-X, 1% IGEPAL, protease inhibitor cocktail (Phos Stop Easy pack Ref:04906837001, Roche) or cold PBS (controls) and incubated in a cold room for 40 minutes. Preextracted sections were washed in PBS and followed by standard antigen retrieval and staining procedure as described in methods section. Primary antibodies against RAD51 (EPR40(30)3, Abcam), ɤH2AX (JBW301, Merck Millipore) and phosphoBRCA1 (ab47276, Abcam) was used for the dilutions 1:400, 1:1000 and 1:150 respectively.

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Supplementary Figure B. RAD51 antibody validation algorithm

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Supplementary figure C. Calu-1 cells; immunohistochemical expression of ɤH2AX (double strand break marker), phosphoBRCA1 and RAD51 (without pre-extraction of abundant RAD51 protein), in non-irradiated (control) and irradiated cells. Magnification x20

Supplementary figure D. Calu-1 cells; For the better visualization of irradiation induced RAD51 foci, by immunohistochemistry, we performed the pre-extraction of abundant RAD51 protein before staining. The remaining RAD51 attached to DNA – ’’RAD51 foci’’ is shown in figures A and B, Magnifications x40 and x100 respectively

Supplementary Figure E. Kaplan-Meier estimates of progression-free survival (PFS) according to

nuclear RAD51 immunoreactivity (threshold for RAD51 loss of less than 0.1) in the replication (panel

A) and discovery set (panel D), as well as in the subgroups of patients receiving perioperative

chemotherapy (panels B, E) and perioperative radiotherapy (panel C). There is no separate Kaplan-Meier

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curve for patients in the discovery set receiving perioperative radiotherapy, because this subgroup did not

contain any patient. Hazard rations (HR) are for loss of nuclear RAD51 immunoexpression. Blue curves

represent tumors positive for nuclear RAD51 immunoexpression, red curves represent tumors with loss of

nuclear RAD51 immunoexpression.

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Supplementary Figure F. Kaplan-Meier estimates of overall survival (OS) according to nuclear RAD51

immunoreactivity (threshold for RAD51 loss of less than 0.1) in the replication (panel A) and discovery

set (panel D), as well as in the subgroups of patients receiving perioperative chemotherapy (panels B, E)

and perioperative radiotherapy (panel C). There is no separate Kaplan-Meier curve for patients in the

discovery set receiving perioperative radiotherapy, because this subgroup did not contain any patient.

Hazard ratios (HR) are for loss of nuclear RAD51 immunoexpression compared to positive nuclear

RAD51 immunoexpression. Blue curves represent tumors positive for nuclear RAD51

immunoexpression, red curves represent tumors with loss of nuclear RAD51 immunoexpression.

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