IGFBP7 knockdown or neutralization decreases ALL cell line proliferation, survival, and migration. (A) Proliferation of REH, RS4;11 (BCP-ALL), TALL-1, and Jurkat (T-ALL) cell lines stably transduced with lentivirus expressing shRNA against IGFBP7 (sh.959) or scramble control (Scr) analyzed by the trypan blue exclusion technique. Results from 3 independent experiments run in triplicate wells, using RPMI-3% FBS. (B) Cell cycle analysis of cells after 4-hour incubation with BrdU, using RPMI-10% FBS. The percentage of cells in G0/G1, S-phase, G2/M, and in apoptosis (sub-G0/G1) was evaluated by flow cytometry. Results representative of 3 independent experiments run in triplicate wells (see supplemental Figure 2A). (C) Cells were cultured in RPMI-3% FBS for 24 hours, and the percentage of apoptotic cells was evaluated by flow cytometry after Annexin-V-FITC/7AAD staining. Bars represent means ± standard error (SE) of 4 independent experiments run in duplicate wells. See also supplemental Figure 2C. (D) Migration of ALL cells in the transwell system toward SDF-1 (100 ng/mL) or PBS 1×, using RPMI-10% FBS. Cells in the lower chamber were counted by flow cytometry after 4-hour migration. Bars represent means ± SE for triplicate wells. (E) Viability of ALL cells measured by the MTT assay after 48-hour culture in RPMI-10% FBS supplemented with an anti-IGFBP7 (clone C311, 20 µg/mL) or anti-PSA control (20 µg/mL) monoclonal antibody. Bars represent means ± SE for 3 independent experiments run in triplicate wells. Statistical analyses were done by 1- or 2-way analysis of variance (ANOVA) and Bonferroni posttests (*P ≤ .05, **P .01, ***P .001, and ****P .0001).