PRO-001 inhibits FGF-mediated ERK1/2 phosphorylation and inhibits the viability of UTMC2 in the presence of FGF, IL-6, IGF-1, and BMSCs. (A) Human myeloma cell lines (HMCLs) expressing FGFR3 (UTMC2, H929, KMS11, KMS18) or not (8226) were stimulated with FGF and incubated with 5 μg/mL PRO-001, control antibody, or 100 nM PD173074. Growth at 48 hours was assessed by MTT assay and is reported as PI = (OD_+FGF±DRUG)/(OD_-FGF), where OD_+FGF±DRUG is the OD in the presence of FGF with or without inhibitor and OD_-FGF is the OD in the absence of FGF. When PI = 1, this indicates no enhanced growth with the addition of FGF. ▪ indicates stimulated with FGF; ▧, FGF plus control antibody; , FGF plus 5 μg/mL PRO-001;, FGF plus 100 nM PD173074. (B) Flow cytometric analyses of ERK1/2 phosphorylation. UTMC2 cells were stimulated with aFGF (black line) or pretreated with 5 μg/mL PRO-001 and then stimulated with FGF9 (gray line). The filled histogram indicates unstimulated cells. (C) UTMC2 cells were cultured in media containing 2.5% FCS and 10 ng/mL FGF9 with control antibody (□), 5 μg/mL PRO-001 (), or 100 nM PD173074 (▧) in the presence or absence of 50 ng/ml IL-6 or 50 ng/ml IGF-I or both. Cell viability after 48 hours was assessed by MTT assay and is reported as PI = (OD_+CYTOKINES)/(OD_-FGF), where OD_+CYTOKINES is the OD in the presence of FGF with or without IL-6 or IGF-I or both and OD_-FGF is the OD in the absence of FGF. When PI = 1, this indicates no enhanced growth with the addition of cytokines. (D) BMSCs alone or BMSCs together with UTMC2 cells (□) were cultured with control antibody () or 5 μg/mL PRO-001 (▧) for 72 hours and apoptosis was assessed by means of flow cytometric assay of annexin V binding and propidium iodide exclusion. Values represent means of quadruplicate cultures ± SD.