Fig. 4

An increase in CXCL13 upon the interaction between MM cells and macrophages is mediated by BTK and TGFβ signaling. A Peripheral-blood-derived MΦ were cultured in the absence or presence of MM cells RPMI8226 (direct co-culture) with or without ibrutinib (20 µM), acalabrutinib (20 µM) and zanubrutinib (20 µM) for 48 h. The levels of secreted CXCL13 in the culture medium were evaluated by ELISA. Data are presented as the mean of triplicates ± STDEV (**p < 0.01). B Peripheral-blood derived MΦ were cultured in the absence or presence of MM cell lines RPMI8226 and CAG, separated by 0.4 µm membrane with or without ibrutinib (20 µM), acalabrutinib (20 µM) and zanubrutinib (20 µM) for 48 h and subjected to subsequent analysis. Expression levels of CXCL13 mRNA in MΦ cells evaluated by qRT-PCR. C MM-induced signaling in MΦ co-cultured in 0.4 µm transwells for 48 h, evaluated by Western blot analysis. Blots were probed for p-c-Jun, p-JNK, p-STAT3 and p-p38. β-actin was used as an internal control. Representative data from at least two independent experiments are shown. D Peripheral-blood-derived MΦ were cultured in the absence or presence of MM cells RPMI8226 (direct co-culture) for 48 h and subjected to flow cytometry analysis. Expression levels of cell surface CD206, CD163, and MERTK on gated CD11b+ cells are depicted in representative histograms. Quantification of geometric mean fluorescence intensity (MFI) of surface markers and percentage of positive cells was performed. E CXCL13 mRNA levels in MM cells RPMI8226 and CAG, cultured in the absence or presence of peripheral-blood derived MΦ, treated with SB-431542 (20 µM) for 48 h, evaluated by qRT-PCR. F Peripheral-blood-derived MΦ were cultured in the absence or presence of MM cells RPMI8226 and CAG (direct co-culture) with or without SB-431542 (20 µM) for 48 h. Levels of secreted CXCL13 in the culture medium were evaluated by ELISA. Data are presented as the mean of triplicates ± STDEV (**p < 0.01)