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. Author manuscript; available in PMC: 2022 Mar 1.
Published in final edited form as: FEBS J. 2020 Sep 25;288(6):1871–1886. doi: 10.1111/febs.15541

Figure 2. ICAM-1 and CCR2 expressed by PIN mediate cell attraction between macrophages and PIN cells.

Figure 2.

(A) A diagram for demonstrating cell co-cultivation of Pr111 cells and Raw264.7 macrophages that were in vivo labeled with Vybrant DiI (Red) and Vybrant DiO (green) respectively. Cells were plated on matrigel in 3D in μ-Slides (Ibidi) and overlaid with PrEBM complete media. Fluorescent and bright field pictures of Pr111 and Raw264.7 cells were taken at 0 h and 72 h of co-culture. Scale bar: 25 μm. n=3. (B) Raw 264.7-coculture media and control media were subjected to a mouse cytokine array for identifying the cytokines present in Raw 264.7-coculture media. A1/2, A23/24, and F1/2 contain positive controls, and F23/F24 contains negative controls. (C-D) Similar to A, Pr111 cells grown on matrigel in 3D were co-cultured with Raw264.7 cells in the presence of isotype control antibody or the neutralizing antibody against the cytokines identified from Raw 264.7-coculture including C5a (C), ICAM-1 and CCL2 (D). The data shown in C-D is representative of 3 independent experiments. Cell attraction between Pr111 and Raw264.7 cells under these treatments were evaluated 72 h post co-culture and quantified. Scale bar: 25 μm. The data was analyzed by one-way ANOVA with Tukey-Kramer post-hoc test. *: p<0.05 as compared to isotype control NAb, n=3. (E-F) Normal prostate and prostate cancer sample containing PIN of human tissues were immunostained for expression of ICAM-1 (E) and CCR2 (F). The figure shown is representative of three human tissue samples. Scale bar: 50 μm.