Fig. 6

CXCL13 silencing in MM cells inhibits MM growth in vivo and suppresses MM-associated osteoclastogenesis in murine BM. A CXCL13 mRNA levels in RPMI8226-CXCR4-GFP and RPMI8226-CXCR4-GFP-CRISPR-CXCL13 cells evaluated by qRT-PCR. B CXCL13 secreted levels in conditioned medium of RPMI8226-CXCR4-GFP and RPMI8226-CXCR4-GFP-CRISPR-CXCL13 cells, cultured alone or co-cultured with peripheral-blood derived MΦ. C Peripheral-blood derived MΦ were cultured in the absence or presence of MM cell lines RPMI8226-CXCR4-GFP and RPMI8226-CXCR4-GFP-CRISPR-CXCL13, separated by 0.4 µm membrane for 48 h and subjected to subsequent analysis. Expression levels of CXCL13 and RANKL mRNA in MΦ cells evaluated by qRT-PCR. D In vitro cell growth kinetics of RPMI8226-CXCR4-GFP and RPMI8226-CXCR4-GFP-CRSIPR-CXCL13 cells, enumerated by FACS. E Transwell migration of RPMI8226-CXCR4-GFP and RPMI8226-CXCR4-GFP-CRSIPR-CXCL13 cells during 4 h in response to CXCL12 (200 ng/mL), evaluated by FACS. F-H NSG mice were i.v. inoculated with RPMI8226-CXCR4-GFP cells (n = 4) or RPMI8226-CXCR4-GFP-CRISPR-CXCL13 cells (n = 4). F MM tumor load was measured by enumeration of GFP+ human MM cells in the BM of inoculated animals. Data are presented as mean ± SE, **p < 0.01. G MNCs from the BM of control (n = 4) and MM-inoculated (n = 5) mice were purified and RNA was extracted. Gene expression of murine factors was evaluated by qRT-PCR. H Murine RANKL levels in plasma of non-inoculated (n = 3), RPMI8226-CXCR4-GFP (n = 4) and RPMI8226-CXCR4-GFP-CRISPR-CXCL13-inoculated (n = 4) animals at day 24 following cell injection. Data are presented as mean ± STDEV, **p < 0.01