Fig 2: IGFBP2 promoted migration and invasion in ESCC cell lines through Akt, Erk, and NFκB signaling pathways. (A) Transwell migration assay of three ESCC cell lines stimulated with recombinant human IGFBP2 (rhIGFBP2). RhIGFBP2 was added to the lower chamber at a concentration of 100 ng/mL. Migrating cells on the bottom of the upper chamber were counted in five randomly selected fields. (B) Transwell invasion assay of three ESCC cell lines stimulated with rhIGFBP2. RhIGFBP2 was added to the lower chamber at a concentration of 100 ng/mL. Invading cells on the bottom of the upper chamber were counted in five randomly selected fields. (C) Western blotting to confirm the changes in the levels of total and phosphorylated Akt, Erk, and NFκB for 0, 10, 30, and 60 min after stimulation with rhIGFBP2 (100 ng/mL). TE-8, TE-9, and TE-15 cells were seeded on the day before protein extraction and incubated overnight in serum-free RPMI-1640. (D,E) Transwell migration (D) and invasion (E) assay of ESCC cell lines with inhibitors of phosphatidylinositol 3-kinase (PI3K inhibitor; LY294002, 10 μM), MEK1/2 (PD98059, 10 μM), and NFκB (Bay117082, 10 μM), stimulated with rhIGFBP2. Data are presented as mean ± SEM (* p < 0.05, ** p < 0.01, *** p < 0.001).

Fig 3: MT2A promotes the expression and secretion of IGFBP2 in CAF-like cells. (A,B) Three types of CAF-like cells (CAF8. CAF9, and CAF15) were transfected with siRNA targeting MT2A (siMT2A) and control siRNA (siNC). The knockdown was confirmed by qRT-PCR (A) and Western blotting (B). After Western blotting, the normalized relative expression fold-change was calculated using the ImageJ software, and the values were arbitrarily set as 1.00 for cells transfected with siNC. (C) Antibody array with monocultured MSC (monoMSC), CAF15, siNC-transfected CAF15 and siMT2A-transfected CAF15 conditioned medium. The intensity of insulin-like growth factor binding protein 2 (IGFBP2) was quantified using the public domain software ImageJ, normalized to a positive control reference spot. (D–F) Expression levels and secretory concentrations of IGFBP2 in CAF8, CAF9, and CAF15 cells were compared with those in MSCs using qRT-PCR (D), ELISA (E), and Western blotting (F). After Western blotting, the normalized relative expression fold-change was calculated using the ImageJ software, and the values were arbitrarily set as 1.00 for control MSCs. (G) Expression of IGFBP2 was detected by qRT-PCR in CAF8, CAF9, and CAF15 cells transfected with siNC and siMT2A. (H) The secretory concentrations of IGFBP2 were detected by ELISA in CAF8, CAF9, and CAF15 cells transfected with siNC and siMT2A. (I) Western blotting for IGFBP2 in CAF8, CAF9, and CAF15 cells transfected with siNC and siMT2A. After Western blotting, the normalized relative expression fold-change was calculated using the ImageJ software, and the values were arbitrarily set as 1.00 for cells transfected with siNC. Data are presented as mean ± SEM (* p < 0.05, ** p < 0.01, *** p < 0.001).

Fig 4: IGFBP-2 silencing in MCF-7 cells with or without IGF-I and etoposide. (A) Shows the mean of percentage cell death. (B) Western blot analysis of cell lysates of MCF-7 cells when IGFBP-2 siRNA is present and absent and 10 ng/mL IGF-I treated with and without etoposide (40 µM) for 23 h. As a loading control, tubulin was used. (C) Optical densities of MCF-7 lysates of treated cells expressed as relative fold change after correcting to tubulin and normalising to 1 (where * p < 0.05, ** p < 0.01 and *** p < 0.001) of three independent experiments each repeated in triplicate. (NS = non-silencing). One-way ANOVA was used for statistical analysis. (D) Nuclear (N) and cytoplasmic (C) fractions of MCF-7 cells were analysed by Western blotting. As nuclear and cytoplasmic loading controls, lamin A/C and tubulin were used, respectively. (E–G) Immunofluorescence staining of IGFBP-2 (green), P-DNA-PKcs (green), and γH2AX (red) in MCF-7 cells. FIJI/Image J was used to process the images (scale bar = 50 μm). Three independent experiments were performed with each repeated in triplicate. (H) Quantification of intensity of immunofluorescence within the nucleus for IGFBP-2, P-DNA-PKcs, and γH2AX in MCF-7 cells. Immunofluorescence images was analysed by Fiji-image J program, which measures the fluorescence intensity of each protein inside the nucleus. Three independent experiments were performed with each repeated in triplicate. Original western blots are presented in File S1.

Fig 5: Immunofluorescence staining of MDA-MB-231 cells in the presence and absence of recombinant IGFBP-2 (250 ng/mL) for 4 h and RGD (0.1 µg/mL) for 1 h, treated with and without etoposide (40 µM) for 19 h (scale bar = 50 μm). (A) Immunofluorescence staining of IGFBP-2 (red), γH2AX (red), and P-DNA-PKcs (green). (B) Quantification intensity of immunofluorescence within the nucleus for IGFBP-2, γH2AX, and P-DNA-PKcs. Results shown are the mean of three independent experiments. Immunofluorescence images were analysed by Fiji-image J program that measures the fluorescence intensity of each protein inside the nucleus (where *** p < 0.001).