FIGURE 3.

IL‐1β derived from macrophages induces CD26 expression in malignant pleural mesothelioma (MPM) cells. A, Schema of coincubation assay. THP‐1‐derived macrophages and H2452 cells were coincubated for 5 d and separately analyzed. B, Expression of transcripts of M1 marker iNOS and M2 marker ARG1 in THP‐1‐derived macrophages in the absence or presence of coincubating with H2452 cells are shown in the left and right panels, respectively. C, Amounts of IL‐1β transcripts in H2452 cells and THP‐1‐derived macrophages were measured by qRT‐PCR. Results of the samples of THP‐1‐derived macrophages in the insert (THP‐1 ins) and H2452 cells in the bottom well (H2452 btm) harvested after coincubation using Transwell (Costar) inserts were presented in the third and fourth lane from the left, respectively. D, ELISA analysis about the concentration of IL‐1β in the culture medium. Samples were collected from the supernatants of H2452 cells, THP‐1‐derived macrophages, and co‐culture medium of these cells. ND, not detected. E, Amount of CD26 transcripts in H2452 cells under coincubation with THP‐1‐derived macrophages was also analyzed by qRT‐PCR. F, Expression of CD26 transcripts in H2452 cells when coincubated with THP‐1‐derived macrophage in the presence of 1 μg/mL of anti‐IL‐1β neutralizing Ab (αIL‐1β) or its isotype control mouse IgG1 (mIgG1) was evaluated. G, CD26 gene expression in H2452 cells was examined when coincubated with either control or CASP1 siRNA‐transfected THP‐1‐derived macrophages. H, Expression of HMGB1 in H2452 cells under coincubation with THP‐1‐derived macrophages was examined by immunoblotting. I, Representative immunofluorescence images are shown of HMGB1 expression in H2452 cells when they were coincubated with THP‐1‐derived macrophages. All the experiments were performed at least 3 times. Quantification data represent mean ± SEM. *P < .05, **P < .01, Scale bars, 50 μm