Journal: Cancer letters

Article Title: NCAM- and FGF-2-mediated FGFR1 signaling in the tumor microenvironment of esophageal cancer regulates the survival and migration of tumor-associated macrophages and cancer cells.

doi: 10.1016/j.canlet.2016.06.009

Figure Lengend Snippet: Fig. 1. Induction of NCAM in peripheral blood monocyte (PBMo)-derived macrophages stimulated with conditioned medium of a TE series ESCC cell line (TECM). PBMos were treated with 25 ng/mL recombinant human M-CSF for 6 d to induce Macrophage_L and then exposed to the TECM (TE-8, TE-9 and TE-15) for 2 d to induce TAM_Ls. (A) NCAM mRNA induction in the TAM_Ls was confirmed by RT-PCR (left panel). The results were normalized to GAPDH as a control, and densitometric analysis of bands was performed with ImageJ (National Institutes of Health, Maryland, USA) (right panel). The results are the mean ± SEM (n = 3, *p < 0.05). (B) NCAM expression levels in the TAM_Ls were determined by quantitative RT-PCR, compared with those of the Macrophage_Ls, and normalized to GAPDH expression. The results are the mean ± SEM (n = 3, *p < 0.05). (C) NCAM induction was also confirmed in the TAM_Ls by western blotting (left panel). The results were normalized to β-actin as a control, and densitometric analysis of bands was performed with the ImageJ (right panel). The results are the mean ± SEM (n = 3, *p < 0.05). (D) NCAM and CD11b expression in Macrophage_Ls and TAM_Ls. Double immunofluorescence was performed using anti-NCAM (green) plus the macrophage marker anti-CD11b (red). NCAM expression was detectable in TAM_Ls_TE-9 (left panel). Nuclei were stained with DAPI (blue). Scale bar, 10 μm. NCAM fluorescent intensity data were quantified using ImageJ (right panel). The corrected total cell flu- orescence (CTCE) was calculated as integrated density −(area of selected cell × mean fluorescence of background reading). The results are the mean ± SEM (n = 3, **p < 0.001). (E) The localization of NCAM and F-actin in TAM_Ls_TE-9. Immunofluorescence was performed using anti-NCAM (green) in TAM_Ls_TE-9. Nuclei and F-actin were stained with DAPI (blue) and phalloidin (red), respectively. NCAM and F-actin were co-localized at a part of the lamellipodia (arrowheads). Scale bar, 10 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The following selective inhibitors and recombinant protein were used: PI3K inhibitor (LY294002, 20 μM; Cell Signaling Technology, Beverly, MA); FGFR1 inhibitor (SU5402, 50 μM; Santa Cruz Biotechnology, Dallas, TX); human recombinant basic fibroblast growth factor (rhFGF-2, 5–20 ng/mL; R&D Systems).

Techniques: Derivative Assay, Recombinant, Reverse Transcription Polymerase Chain Reaction, Control, Expressing, Quantitative RT-PCR, Western Blot, Marker, Staining