TSP-1 and its membrane receptors' expression in NB4 and NB4-LR1 cell lines. Maturation-sensitive NB4 cells and maturation-resistant NB4-LR1 cells were cultured for 4 days either untreated or treated with 1 nM to 1 μM ATRA or 1 μM ATRA and processed for examination of TSP-1 transcript (A, C) and protein level expression (B) or TSP-1 receptors' membrane expression (D). (A) Cell maturation was monitored by the NBT dye reduction assay. Total RNA (20 μg) was hybridized with a ³²P-labeled fragment of human cDNA for TSP-1. Equivalent loading and integrity of each RNA preparation were evaluated by ethidium bromide staining of the 28S rRNA bands. (B) Triton X-100 cell lysates were prepared from maturation-sensitive NB4 cells and processed for immunoprecipitation and Western blotting for analysis of TSP-1 protein expression, as described in “Materials and methods.” Cell secretion of TSP-1 into the extracellular medium was similarly analyzed upon Triton X-100 solubilization of the corresponding culture media. Here, Triton X-100 cell extract and Triton X-100-solubilized culture medium coming from 4 × 10⁶ NB4 cells and 1.2 × 10⁵ NB4 cells, respectively, were analyzed. (D) Untreated (dotted line) or ATRA-treated (1 μM; bold solid line) NB4 and NB4-LR1 cells (2 × 10⁵) were incubated with 20 μg/mL monoclonal antibodies to CD36, CD47, the β1 or β3 integrin subunit, or 20 μg/mL isotype-match control antibody (thin solid line). Alexa Fluor 488-conjugated anti-mouse IgG was used as secondary antibody (1:700), and cells were analyzed by flow cytometry.