ATX inhibitor-S32826 induced changes in cultured human TM cells.

<p>A. Primary cultures of human TM cells cultured either on gelatin-coated glass coverslips or in plastic petridishes were treated with S32826 (10 µM) in the absence of serum or in the presence of 0.5% fetal bovine serum for 24 hrs. Based on phase contrast microscopic examination, treatment of TM cells cultures with drug caused the appearance of membranous floating clumps (arrows) in culture medium, without any noticeable changes in cell morphology and cell adhesion relative to control cells treated with DMSO vehicle. This response appeared to increase progressively with time. B. The membranous floating material generated from drug (S32826) treated cells (A) was extracted from the cell culture media by centrifugation, rinsed with PBS, dissolved in Laemmli sample buffer, separated by SDS-PAGE and stained with gelcode blue as shown in the figure (two independent samples are shown from each treatment). Subsequently, the predominant protein bands were gel extracted and subjected to mass spectrometry-based identification. This analysis revealed presence of proteins involved in lysosomal exocytosis, trafficking and membrane repair as discussed in the results section.