The prosurvival effect of IGFBP7 is insulin (IGF) dependent. (A) ALL cell lines were cultured in RPMI-10% FBS supplemented with insulin (500 ng/mL) and/or IGFBP7 (100 ng/mL) for 48 hours. Cell viability was quantified by the MTT assay. Bars represent means ± SE for 2 independent experiments run in triplicate wells. (B) Proliferation of RS4;11 and Jurkat cell lines expressing shRNAs against IGFBP7 (sh.959) or scramble control (Scr) analyzed by the trypan blue exclusion technique after insulin (500 ng/mL) and/or IGFBP7 (100 ng/mL) treatment. Results from a single experiment run in triplicate wells, using RPMI-3% FBS. See also supplemental Figure 3. (C) Primary BCP-ALL cells after 48 hours or (D) primary T-ALL cells after 24 hours cultured in AIM-V serum-free medium supplemented with insulin (500 ng/mL) and/or IGFBP7 (100 ng/mL). Cell viability was measured by Annexin-V/7AAD staining and flow cytometry. Bars represent means ± SE of Annexin-V/7AAD negative fraction of 2 independent experiments run in duplicate wells. See also supplemental Figure 4A. (E) Primary BCP or T-ALL cells were cultured in AIM-V serum-free medium (Control) supplemented with an anti-PSA control (20 µg/mL) or anti-IGFBP7 antibody (clone C311, 20 µg/mL) for 48 or 24 hours, respectively. Apoptosis was measured by Annexin-V/7AAD staining and flow cytometry. See also supplemental Figure 4B. High IGFBP7 concentrations (20 µg/mL) are detrimental to primary BCP- and T-ALL (F) cells after 24 hours of treatment in AIM-V serum-free medium supplemented with insulin (500 ng/mL). See also supplemental Figure 4C. Statistical analysis was done by 1- or 2-way ANOVA and Bonferroni posttests (*P .05, **P .01, ***P .001, and ****P .0001).