Human splenic fibroblasts reduce BCL-2 dependence in ETP-ALL in vitro. (A) Schematic representation of coculture system using HSF and LOUCY cells. (B) BH3 profile of LOUCY cells cocultured with HSF for 48 hours (mean ± standard deviation [SD]; n = 3). (C) Percentage of surviving cells after 24 hours of ABT-199 treatment following a 48-hour coculture with HSF and the subsequent 50% inhibitory concentration (IC₅₀) values listed (mean ± SD; n = 3); 2-way analysis of variance with post hoc Dunnett’s comparison. (D) Western blot analysis of antiapoptotic BCL-2 proteins (BCL-2, BCL-XL, and MCL-1) and BIM protein expression in LOUCY and CC_HSF for 24 and 48 hours. β-actin was used as loading control (n = 3). (E) The mRNA expression of BCL-2, BCL2L, MCL-1, and BCL2L11 in CTRL and CC_HSF 24 h and 48 hours was measured by using quantitative reverse transcription polymerase chain reaction. The mRNA gene expression was normalized to GAPDH. Mean of n = 3, error bars show ± SD. (F) Transwell migration assay of LOUCY toward the condition media generated from HSF and HS-5 (bone marrow stroma cell line). Representative microscope pictures in bright field and Calcein-AM of LOUCY after 48 hours of migration toward the CM and the migration ratio (mean ± SD; n = 3). One-way analysis of variance statistical test was performed to determine the P values. The bar scale shows 100 μm. Relative ratio of migrating cells to migrating control cells was determined. Plotted as the mean ± SD of 3 independent experiments. (G) Percentage of surviving ETP-5 cells after 6 hours of ABT-199 and ABT-263 treatment and the subsequent IC₅₀ values listed. (H) BH3 profile of ETP-5 cells cocultured with HSF for 16 hours. *P ≤ .05, **P ≤ .01, ***P ≤ .001. ns, not significant.