Effects of ATRA on PLSCR1 expression in NB4 cells. NB4 cells were treated with the indicated concentrations of ATRA for 3 days (A-D), or with 10^-6 M ATRA for days shown (E-G). CD11b⁺, CD11c⁺, and/or CD14⁺ cells (A,E) were measured by flow cytometry as described in “Materials and methods.” PLSCR1 protein (B,F) was detected by Western blot with β-actin as loading control. PLSCR1 mRNA was detected by real-time quantitative PCR and semiquantitative RT-PCR. (C) Typical amplification plots for PLSCR1 and β-actin showing how their relative expression levels can be assayed by real-time RT-PCR using cDNA template derived by reverse-transcriptase treatment of RNA from a single sample. Upper panel shows that amplification plots for β-actin in different multiplex tubes used to assay PLSCR1 are closely superimposed within experimental error. Middle panel shows amplification plots for PLSCR1 in untreated control, and samples treated with 10^-5 M or 10^-8 M ATRA; the difference between these assays at the cycle threshold detection line represents the ΔΔCt value when the β-actin results superimpose exactly. Lower panel shows the PLSCR1 mRNA level detected by real-time quantitative PCR under the indicated concentrations of ATRA. For semiquantitative RT-PCR (D,G), the signal intensities of amplified PLSCR1 fragments were normalized against 983-bp G3PDH using a densitometer. Each point represents the mean from triplicate samples with a variance of less than 15%. All experiments were repeated at least 3 times with similar results.