Fig 2: aFGF enhances SCs proliferation and migration.a Co-immunofluorescence images showed the distribution of Ki67+ (green) labeling nucleus were colocalized with S100 (red) in the control, T2DM and T2DM + aFGF groups. Scale bar equals 100 µm. b Quantification of Ki67 positive cells/100 mm2 in each group. n = 7 animals per group. c-e Western blotting analyzed the level of PCNA and Ki67 in each group (n = 3). GAPDH was used as an internal loading control and all protein expression was shown as relative levels to that of the control. f–h The expression of PCNA and Ki67 proteins was quantified in SCs of control, HG and HG + aFGF groups (n = 3). i–j Immunofluorescence staining and quantification data of Ki67 (green) and S100 (red) signals in the cell level. Nucleus was stained with DAPI. Scale bar = 25 µm. k, l SC migration analysis was detected through a wound-healing assay at 0 and 12 h after scratch. n = 3, scale bar = 100 µm. Data are the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the control group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. the T2DM group or HG group.

Fig 3: CRC-secreted miR-25-3p primes pre-metastatic niche. a Effects of NCM460 exosomes, SW480/mock exosomes, SW480/miR-25-3p exosomes, SW480/miR-25-3p exosomes + miR-25-3p inhibitor treatments on vascular permeability of mice liver by in vivo permeability assay. The mice were injected with rhodamine–dextran after exposure to PKH67-labeled exosomes. Levels of rhodamine–dextran fluorescence in tissues were quantified using Image J software and normalized to the levels of DAPI. Mean ± SEM are provided (n = 5). Scale bar represents 50 µm. b Effects of NCM460 exosomes, SW480/mock exosomes, SW480/miR-25-3p exosomes, SW480/miR-25-3p exosomes + miR-25-3p inhibitor treatments on vascular KLF4, KLF2, and ZO-1 expression (red) in hepatic vessels by immunofluorescence. The vascular structures were labeled by CD34 (green). Scale bar represents 50 µm. c Effects of NCM460 exosomes, SW480/mock exosomes, SW480/miR-25-3p exosomes or SW480/miR-25-3p exosomes + miR-25-3p inhibitor treatments on KLF4, ZO-1, occludin, Claudin5, KLF2, VEGFR2, fibronectin, and S100 expression in mice liver by Western blot. d The mice were intra-spleen injected with naked SW480 cells after exposure to NCM460 exosomes, SW480/mock exosomes, SW480/miR-25-3p exosomes or SW480/miR-25-3p exosomes + miR-25-3p inhibitor treatments. The number of liver metastatic sites (indicated by arrows) was counted under the microscope. Mean ± SEM are provided (n = 5). Scale bar in left panels represents 0.5 cm. Scale bar in right panels represents 100 µm. **P < 0.01, ****P < 0.0001 according to two-tailed Student’s t test

Fig 4: Immunohistochemistry and immunofluorescence of the gap junction protein connexin 43 and the satellite cell marker S100 in DRG of normal controls and FA. a-d normal control; e-h FA; a and e positive contrast immunohistochemistry of connexin 43; b and f Alexa 488 immunofluorescence of S100; c and g Cy3 fluorescence of connexin 43; d and h, merged images of b and c, and f and g, respectively. In the normal DRG, connexin 43 shows punctate and coalescing reaction product (a; and inset [arrow]). In FA, a great abundance of connexin 43 reaction product is present in multiple layers around small neurons (e). Hyperplastic satellite cells bridge gaps between neurons. The confocal images of a normal DRG show sparse gap junctions by their punctiform connexin 43 immunofluorescence (c). They are closely related to S100-positive satellite cells (d). In FA, the number of connexin 43-reactive puncta is greatly increased (g and h). Bars: (a) and (e), 20 µm; insets, 10 µm; (b-d) and (f-h), 10 µm. N, neuron

Fig 5: Immunohistochemistry of glutamine synthetase, S100, and laminin in DRG of normal controls and FA. a-c normal controls; d-f FA; a and d glutamine synthetase; b and e S100; c and f laminin. In normal DRG, reaction product of glutamine synthetase (a) and S100 (b) reveal well organized layers of adjacent and overlapping satellite cells around each neuron. Normal nerve cells are surrounded by delicate layers of laminin (c). In FA, the remaining neurons are smaller, and satellite cell layers are thicker (d and e, insets) and disrupted (d, inset). Laminin reaction product shows multiple layers surrounding neurons, which is consistent with proliferation of satellite cell processes (f, inset). The arrows in (d) and (e) point to residual nodules that are reactive with antibodies to glutamine synthetase (d) and S100 (e), respectively. Bars: (a-f), 100 µm; insets, 20 µm