Figure 7.

FeAS affects the expression of adhesion molecules on MS5 stroma cells and alters the profile of cytokine production. (A) After culture with or without FeAS, MS5 were stained as indicated and analyzed by fluorescence microscopy. Total ROS were detected with Red CC-1, intracellular labile iron with Calcein-AM, and nuclei with Hoechst 33,342. (B) After MS5 cells were cultured with or without FeAS for 48 h, cells were stained with the indicated Abs. Representative flow cytometry histograms from three independent experiments are shown. (C) Conditioned media were analyzed with a cytokine array and the accompanying kit reagents and protocol. Conditioned media from FeAS-treated (10 or 50 μM) or untreated MS5 cells (CTL) was collected after 48 h. of culture. Medium supplemented with 10% FBS αMEM was used as a negative control (NC). The assay is arranged in a 14 × 10 grid (Array 3) and a 12 × 8 grid (Array 4), with the configuration shown in Supplementary Data (Figure S1A,B). Summarized densitometry data (mean ± SD) from three independent experiments is shown in the right panel (* p < 0.05). (D) MS5 cells were cultured in the presence or absence of FeAS for 48 h, and co-cultured with LSKs with or without FeAS for another 48 h and the number of cobblestone-like areas seen in each well were counted under light microscopy. Summarized data (mean ± SD) from two independent experiments is shown (* p < 0.05). Unt-FeAS: FeAS was present in the culture medium only when MS5 cells were co-cultured with LSK; FeAS-CTL: FeAS was present in the culture medium when MS5 cells were cultured alone but not when they were co-cultured with LSK; FeAS-FeAS: FeAS was present in the culture medium in both, when MS5 cells were cultured alone and when they were co-cultured with LSK cells; and Unt-CTL: FeAS was not present in the culture medium neither when MS5 cells were cultured alone nor when they were co-cultured with LSK.