Fig. 4.

Early ATRA-induced transcription factors are not upstream of nSMase2. A: MCF7 cells were seeded in 60 mm dishes and stimulated with vehicle (DMSO) or ATRA (1 μM) for 3 h. RNA was extracted, converted to cDNA, and used as template for the Transcription Factor qRT-PCR array. Data are shown as fold change in ATRA stimulated cells compared with vehicle, n = 1. B: MCF7 cells were transfected with AStar or Sox9 siRNA (20 nM) for 48 h prior to stimulation with vehicle (DMSO) or ATRA (1 μM) for 24 h. Protein was extracted and analyzed for nSMase2 and Sox9 levels by immunoblot using actin as loading control; RNA was extracted and analyzed for nSMase2 by qRT-PCR. Immunoblot is representative of n = 3 (* P < 0.05, n ≥ 3). C: MCF7 cells were transfected with AStar or HoxA5 siRNA (20 nM) for 48 h prior to stimulation with vehicle (DMSO) or ATRA (1 μM) for 24 h. Protein was extracted and analyzed for nSMase2 and HoxA5 levels by immunoblot using actin as loading control. Immunoblot is representative of n = 3. D: MCF7 cells were transfected with AStar or IRF1 siRNA (20 nM) for 48 h prior to stimulation with vehicle (DMSO) or ATRA (1 μM) for 24 h. Protein was extracted and analyzed for nSMase2 and Sox9 levels by immunoblot using actin as loading control. Immunoblot is representative of n = 3. E: MCF7 cells were transfected with AStar or ID1 siRNA (20 nM) for 48 h prior to stimulation with vehicle (DMSO) or ATRA (1 μM) for 24 h. RNA and protein were extracted and analyzed for ID1 (RNA) and nSMase2 (protein) levels by qRT-PCR and immunoblot respectively. Actin was used as reference gene and loading control. Immunoblot is representative of n = 3 (* P < 0.05, n = 3).