Fig 2: FGFR4-mediated radiation response involves regulation of RAD51Proteins were isolated at the indicated time points after irradiation from cultures treated with siRNA targeting FGFR4 (A) or with the FGFR-inhibitor PD173074 (B). The upper panels show typical western blots of RAD51 protein expression in irradiated HT29 cells. The lower panels depict the quantification of RAD51 protein expression from 3 independent experiments normalized to control. (C) RAD51 was overexpressed using a RAD51 expressing vector in HT29 cells as confirmed by western blotting (upper panel). HT29 cells seeded into 6 well plates were transiently transfected with a vector expressing RAD51 or the control vector. For determination of radiation response, cells were co-transfected with RAD51 or control vectors together with either scrambled or FGFR4 siRNAs, before exposure to a single 6 Gy dose of γ-rays. The surviving fraction of the transfected cells was measured by quantification of colonies (lower panel). (D) Representative images of the clonogenicity assay.

Fig 3: RAD51-dependent HR is a crucial mediator of HT29 survival after irradiation(A) Immunofluorescence of RAD51 and γ-H2AX foci formation post IR. HT29 cells were seeded onto cover slips and treated with a single 6 Gy dose of γ-rays. 24 h after IR, cells were formalin fixed, permeabilized, and stained with RAD51 and γ-H2AX antibodies. (B) Western blots showing the effect of RAD51 knockdown on the damage marker, γ-H2AX. (C) Western blots confirming the efficiency of the two tested RAD51 siRNAs. (D) Clonogenic surviving fractions of scrambled/RAD51 siRNA treated HT29 cells showing increased cell killing and induced radiosensitivity of the radioresistant HT29 cells after RAD51 knockdown. Cells were exposed to a single 6 Gy dose of γ-rays, and the surviving fractions were calculated by dividing the number of colonies counted by the corresponding number of cells seeded as described in “Materials and Methods.”

Fig 4: Distribution of staining-based FGFR4 and RAD51 expression in pre-neoadjuvant rectal cancer biopsiesRepresentative staining of biopsies exhibiting negative, weak, moderate and strong FGFR4 (A) or RAD51 (B) staining. Rectal cancer tissues were classified according to overall staining intensity for FGFR4 (C) and RAD51 (D), based on slide scans and morphometric analysis. Scale bar = 50 μm.

Fig 5: FGFR4 expression is upregulated after irradiation in a dose-dependent manner together with key homologous recombination-related proteinsExpression of (A) FGFR4, (B) RAD51, (C) BRCA1 and (D) BRCA2 genes were determined by qPCR, 24 h after exposure to different doses of γ-radiation (0, 2, 4 and 6 Gy) in HT29 cells. The expression levels were calculated relative to GAPDH. (E) Cell cycle distribution of HT29 cells irradiated with a single 6 Gy dose of γ-rays. Analysis was done using FACS at 24, 48 and 72 hours post irradiation. (F) Western blot of cdc2 phosphorylation (Tyr-15) status, Cyclin B1 expression, and the histone H3 phosphorylation (Ser-10) in HT29 cells at different time points after exposure to 6 Gy dose. Beta actin was used as loading control.