AML-derived FABP4 is crucial for blast survival in vivo. (A) FABP4 gene expression (expressed in log2 RPKM values) was obtained from GSE49642 and GSE48846 for nonmalignant CD34+ cells, 22 blood AML, and 21 BM AML patient samples. P value was obtained by Wilcoxon rank-sum test. Middle band denotes the median value with lower and upper bands denoting the first and third quartiles, respectively. (B) AML blasts were cultured alone or with adipocytes or BMSC for 48 hours before the AML were assessed for FABP4 mRNA expression using RT-PCR (n = 6). Data are represented as mean ± standard deviation. (C) Primary AML blasts were infected with FABP4 shRNA1 and shRNA2 and control shRNA, and after 96 hours, were subsequently cultured on adipocytes or BMSC for a further 72 hours. AML blasts counted using flow cytometry and Trypan blue exclusion (n = 4). Data are represented as mean ± standard deviation. (D) Primary AML were infected with FABP4-targeted shRNA1 or control shRNA lentivirus, and after 96 hours, were cultured with adipocytes preloaded with fluorescent FA DAA for 24 hours. AML blasts were analyzed for fluorescence using flow cytometry. (E) Hoxa9/Meis1-transformed cells (1 × 105/mL) were cultured as normal media or normal media with IL-3, IL-6, and SCF supplemented or cocultured on BMSC with normal media or on adipocytes with normal media. Hoxa9/Meis1-expressing cells were counted using flow cytometry and Trypan blue exclusion (n = 4). Data are represented as mean ± standard deviation. (F) Hoxa9/Meis1-expressing cells were infected with mouse FABP4 shRNA or control shRNA, and after 72 hours, analyzed for FABP4 protein expression using western blotting. Blots were reprobed for β-actin to show equal sample loading. (G) Hoxa9/Meis1-expressing cells were infected with FABP4-targeted shRNA or control shRNA, and after 72 hours, incubated either alone, with cytokines, with BMSC, or with adipocytes. Hoxa9/Meis1-expressing cells counted using flow cytometry and Trypan blue exclusion (n = 4). Data are represented as mean ± standard deviation. (H) Hoxa9/Meis1 expressing cells were infected with FABP4-targeted shRNA or control shRNA lentivirus, and after 72 hours, incubated for 24 hours with adipocytes preloaded with fluorescent FA DAA. Hoxa9/Meis1-expressing cells were analyzed for fluorescence using flow cytometry (n = 4). (I) Kaplan-Meier survival curves for C57BL/6 mice injected with Hoxa9/Meis1 FABP4-KD cells or Hoxa9/Meis1 AML control-KD cells. *P < .05; **P < .01. PB, peripheral blood.
