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: METi‐induced modulation of selected phosphorylations is time‐dependent and partially ATM‐dependent. (A) Impact of METi pretreatment duration (6 or 16 h prior to IR) and ATM activity (ATM was inhibited 6 or 1 h prior or 1 h post‐IR) on phosphorylation of MET, ATM, NUMA1, H2AX, CHEK1, and TIF1B, on cleavage of caspase‐8 and on CHEK1 protein levels. Cells were harvested 2 h post‐IR, and β‐actin was used as a loading control. (B) METi‐ and ATMi‐based modulation of cellular phosphorylations measured by SRM in EBC‐1 cells. Listed assays have been monitored in samples treated with ATMi, METi, IR, or the combinations (METi + IR; ATMi + METi + IR). Here are presented the results from statistical comparisons addressing three questions: Which are the significant changes in response to IR alone, compared with control (left); which are the significant changes in response to the combination METi + IR, compared with IR alone (right); and which are the significant changes in response to the combination ATMi + METi + IR, compared with METi + IR (middle). Log2FC (n = 3) is represented by a color code; adjusted P‐values by symbol: <0.0005, ‘***’, <0.005, ‘**’, <0.05, ‘*’. (C) Log2FC per condition is presented in comparison with control sample (untreated) for selected phosphorylation events in EBC‐1 cells upon IR (1 or 8 h) with or without METi and/or ATMi pretreatment as compared to untreated condition (n = 3).

Fig 4: 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.

Fig 5: nIGF1R is associated with nuclear proteins, including NuMA, as identified by IP-MS. (A) Diagram showing the experimental workflow. Nuclear proteins were extracted and immunoprecipitated with an anti-IGF1R antibody. Pull-down proteins were separated with SDS-PAGE, in-gel digested and subjected to MS analysis. The readouts were then bioinformatically processed for nIGF1R interactome generation. (B) The SW480 colorectal cancer cell line was infected with IGF1R-coding lentivirus and selected with antibiotic (puromycin). One cell clone that overexpressed IGF1R was named SW480-OE and used for subsequent IP-MS. Western blot analysis detected a significantly higher IGF1R expression in SW480-OE cells compared with untransfected SW480 cells. GAPDH was used as the loading control. (C) Immunoprecipitated nuclear proteins were separated by SDS-PAGE and visualized using a Colloidal Blue Staining kit (left lane). The whole gel lane was cut into eight pieces according to molecular mass and subjected to MS. Normal mouse IgG was used to replace the anti-IGF1R antibody as the negative control (right lane). The corresponding eight areas in the gel lane were cut, MS-processed and used as background control in bioinformatics analyses. (D) IP-MS-identified nIGF1R interactome ranked by Score (defined as sum ion score of identified protein peptide by IP-MS) and enrichment ratio. The upper bar chart shows the top 10 proteins interacting with nIGF1R. The bottom bar chart shows the top 10 DSB-related proteins interacting with nIGF1R. *Protein discovered in the IP group but not the IgG group. nIGF1R, nuclear insulin-like growth factor 1 receptor; NuMA, nuclear mitotic apparatus protein; IP-MS, immunoprecipitation-coupled mass spectrometry.