Fig 2: METi‐ and IR‐related phosphorylations validated by targeted proteomics. (A) Phosphorylation sites selected for analysis by targeted proteomics. Phosphorylation sites identified in discovery screen (orange squares), phosphorylation sites on proteins identified in the discovery screen (yellow squares), and phosphorylation sites selected from literature (green squares) are linked to their known (full edges) or predicted (dashed edges) kinases. (B) METi‐based modulation of cellular DDR monitored by SRM. Assays were monitored in samples treated with METi, IR, or combination of both. Here are presented the results from statistical comparisons addressing two questions: Which are the significant changes in response to IR alone, compared with control; which are the significant changes in response to the combination of METi and IR, compared with IR alone. Log2FC (n = 3) is represented by a color code; adjusted P‐values by symbol: <0.0005, ‘***’; <0.005, ‘**’; <0.05, ‘*’. (C) Log2FC with standard error bars per condition is presented for selected phosphorylations in comparison with control sample (untreated) within each cell line (n = 3). Note the significant difference between IR alone and the combination for NUMA1 S395, ACIN1 S243 in selected time points and cell lines, as opposed to NBN S343.

Fig 3: Colocalization of NuMA and 53BP1 in the cell nucleus predicts poor prognosis in patients with colorectal cancer. (A) Survival analysis based on TCGA. No significant association between the expression of IGF1R, 53BP1 or NuMA and overall or disease-free survival in patients with colorectal cancer was identified. (B) Representative microphotographs (magnification, ×40) from NuMA-53BP1 and IGF1R-NuMA brightfields in situ PLA. Brown dots mark the colocalizations. (C) Survival analysis for IGF1R-NuMA (left panel) and 53BP1-NuMA (right panel) colocalization in patients with colorectal cancer. Strong nuclear 53BP1-NuMA colocalization was associated with poor survival (P<0.001; low-rank test). NuMA, nuclear mitotic apparatus protein; 53BP1, NuMA-p53-binding protein 1; IGF1R, insulin-like growth factor 1 receptor; TCGA, The Cancer Genome Atlas; PLA, proximity ligation assay.

Fig 4: The relationship between METi and DNA damage‐related phosphorylations in vivo. IHC assessment in a xenograft tumor model of EBC‐1 cells. Animals were treated by oral administration of METi (15 mg·kg−1·day−1 for six consecutive days), local irradiation [a single dose (6 Gy) on day 3], or the combination of these two modalities. Following these treatments, the levels of mentioned proteins or phosphorylations sites were assessed in tumor tissues. Representative images (left panel) and quantification [% of positively stained area; mean (n = 3) ± SEM; right panel] of (A) MET Y1234&5, NUMA1 S395, (B) ATM S1981, SMC3 S1083, and (C) CHEK1 and CHEK1 S345 in EBC‐1 subcutaneous xenografts following IR, METi, or their combination. Statistical analysis was performed with the GraphPad Prism Software using one‐way ANOVA test (*P < 0.05; **P < 0.01; ***P < 0.001).

Fig 5: nIGF1R interacts with NuMA and facilitates NuMA-53BP1 colocalization in response to IR. (A) Candidate peptides of NuMA, from which theoretical spectra were sequentially generated and compared against experimental spectra identified by IP-MS. (B) Co-IP with anti-IGF1R antibody validated nIGF1R colocalization with NuMA in SW480 colorectal cancer and R+ (R-overexpressing IGF1R) cell lines (upper panel). Co-IP with anti-53BP1 antibody validated 53BP1 colocalization with NuMA and IGF1R in SW480 and R+ cell lines (lower panel). IP antibodies were replaced with normal mouse IgG as the negative control. (C) In situ PLA validated nIGF1R colocalization with NuMA using anti-IGF1R and anti-NuMA antibodies in SW480 (upper panel) and R+ (lower panel) cell lines. Red fluorescence dots (arrows) indicate the colocalizations. Either primary antibody or both were removed from the experiment to generate negative controls (first three images). Cell nuclei were stained with DAPI (blue). (D) In situ PLA showed that NuMA-53BP1 colocalization (red dots) was increased in response to IR (2 Gy) in R- (MEF igf1r-/-) cell line but remained unchanged in the R+ cell line. Cell nuclei were stained with DAPI (blue). (E) Dynamic change of NuMA-53BP1 colocalization in response to IR (2 Gy) in R+ and R- cell lines at 30 min, and 1, 2 and 4 h after treatment. Number of NuMA-53BP1 foci represents the average in situ PLA signal per cell from at least 50 cells in each condition. *P<0.05. nIGF1R, nuclear insulin-like growth factor 1 receptor; NuMA, nuclear mitotic apparatus protein; 53BP1, NuMA-p53-binding protein 1; IP-MS, immunoprecipitation-coupled mass spectrometry; IR, ionizing radiation; PLA, proximity ligation assay; R+, IGF1R-positive; R-, IGF1R-negative; MEF, mouse embryonic fibroblast.