EGF promoted myeloma cells clonogenicity. (A) Imaging of colony-forming assays using RPMI8226 cells (CTR-KD and AL-KD) in the presence of EGF neutralizing antibody (+, EGF neutralizing antibody 10 μg/mL; ++, EGF neutralizing antibody 30 μg/mL). The results are quantified in panel B. (C) Colony-forming assay and (D) statistic result of RPMI8226 (CTR-KD and AL-KD) cells in the presence of recombinant EGF (CTR, without rEGF; +, rEGF 10 ng/mL; ++, rEGF 20 ng/mL). (E) RPMI8226 cells were cultured in CTR-M or BMSC-M in the presence of EGF-neutralizing antibody (10 μg/mL) for 48 hours. The EGFR activation was examined by western blotting for pEGFR (Y1173). The results are quantified in the right panel. (F) EGF concentration in BMSC-M (BMSCs from 3 healthy individuals) and BM aspirations from patients with MM (8 patients) was examined by enzyme-linked immunosorbent assay. (E) Two-tailed Student t test or (B,D) 1-way ANOVA with multiple comparisons. *P < .05, **P < .01.