ATRA induces expression of CCLs in NB4 cells. NB4 cells were cultured with ATRA (10−6 M) for 0, 4, 8, 24, 48, 72, and 96 hours. (A) CCL mRNA expression (mean ± SD of 3 independent experiments) was determined at the indicated time points by quantitative PCR. Expression, relative to GAPDH, is plotted. (B) CCL protein levels (mean of 2 independent experiments) were determined in supernatant of NB4 cells at the indicated time points using ELISA. (C) The effect of ATRA on chemoattraction of normal WBCs toward the supernatant of ATRA-stimulated NB4 cells. The percentages of migrated WBCs (mean ± SD of 4 independent experiments) were determined before and 8, 24, 72, and 96 hours after ATRA stimulation of NB4 cells, using a transwell system. *P < .001, **P < .05 compared with supernatant of untreated NB4 cells. (D) Direct regulation of CCL2 and CCL24 by ligand-activated retinoic acid receptors in NB4 cells. NB4 cells were cultured with ATRA alone (10−6 M), with cycloheximide alone (50 μg/mL), or with the combination of ATRA and cycloheximide for 0, 2, 4, and 8 hours. mRNA expression of CCL2 and CCL24 (top panels, mean ± SD of 3 independent experiments) was determined at the indicated time points by quantitative PCR. Expression, relative to GAPDH, is plotted. Protein levels of CCL2 and CCL24 (bottom panels) were measured in supernatant of NB4 cells before and 8 hours after treatment with ATRA and/or cycloheximide using ELISA. ND indicates not detectable; and MDD, minimal detectable dose, according to the manufacturer.
