Fig 2: Effect of IGF1R and INSR inhibition on UGT2B15 gene expression. (A) MCF7 cells were treated with the selective IGF1R inhibitor AEW541 (10 mM) for 48 h (or left untreated, C), after which cells were harvested, total protein was prepared, and IGF1R, UGT2B15, and p53 levels were measured by Western blots. HSP70 levels were measured as a loading control. The bar graph denotes UGT2B15 and IGF1R levels in control (solid bars) and AEW541 treated cells (striped bars). (B) T47D cells were treated with the INSR inhibitor S961 (100 nM and 1 mM) for 2 h. Cells were then harvested and levels of phospho- and total-INSR, UGT2B15, and p53 levels were measured by Western blots. A value of 100% was given to control, untreated cells. * p < 0.01.

Fig 3: IGF1R signal partially induce nuclear localization of FoxM1. (A, B) HeLa S3 cells were cultured with FBS-free medium for 24 h and then incubated in the presence of 0.1 µg/mL IGF1 for 24 h. The cells were fixed and stained for FoxM1 (green) and DNA (red). (A) Representative images are shown. Scale bar, 50 µm. (B) The fluorescence signals were analyzed by an image analyzer. Fluorescence intensities of nuclear FoxM1 in individual cells were plotted with the mean ± SD calculated from one experimental result (n > 930 in each experiment). Two independent experiments were performed and similar results were obtained. The asterisk indicates significant differences using Welch’s t-test. ** p < 0.01. (C) HeLa S3 cells were starved for 24 h and then treated with 100, 200, and 400 ng/mL of IGF1 for 3 h. Whole-cell lysates were prepared and analyzed by western blots with the indicated antibodies. (D) HeLa S3 cells were transfected with non-targeting siRNA (siCtrl) and siRNAs targeting IGF1R (#1, #2). After 48 h, cells were fixed with formaldehyde and staining for FoxM1 (green) and DNA (red). Cells were classified into three categories: (N > C), fluorescence in the nucleus is higher than that in the cytoplasm; (N = C), similar fluorescence intensity throughout the nucleus and cytoplasm; (N < C), fluorescence in the nucleus was lower than that in the cytoplasm. Results represent mean ± SD from three independent experiments (n > 233 in each experiment). The asterisk indicates significant differences using a Tukey–Kramer test. ** p < 0.01, NS, not significant. (E) HeLa S3 cells were treated with or without U0126 in serum-free culture medium containing 100 ng/mL of IGF1 for 24 h, fixed and stained for FoxM1 (green) and DNA (red). Scale bar, 20 µm.

Fig 4: 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.

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.