Fig 1: TNFα stimulation upregulates the FasR protein and caspase 3 mRNA expression levels in RPTEC, but the cells undergo apoptosis only in the presence of exogenous FasL. FasL interaction with upregulated Fas receptor (FasR) is required to induce apoptosis in RPTEC stimulated with TNFα. In TNFα-stimulated RPTEC, caspase 3 and 7 mRNA were significantly upregulated, the mRNA level of caspase 9 did not changed while Bcl2 were significantly downregulated. Additional treatment with 125 ng FasL resulted in upregulation of caspase 3 and caspase 7 mRNA levels and further downregulation of Bcl2 mRNA levels (A). Notably, activated caspase 3 was not detected in unstimulated RPTEC (B, lane 1), RPTEC stimulated with TNFα (B, lane 2) as well as in RPTEC stimulated with TNFα and treated with FasL (B, lane 3). Controls 1 and 2 were cytochrome c treated Jurkat cells (activated caspase 3 positive, II) and untreated Jurkat cells (activated caspase 3 negative, I), lane C1 and C2 respectively. Caspase GLO 3/7 Assay were performed in RPTEC with TNFα stimulation alone and with TNFα stimulation in the presence of FasL. The results are represented from three individual experiments in relative light units (RLU) and values were subtracted from blank (media without cells) (C). In adherent unstimulated or TNFα-stimulated RPTEC, no cells were TUNEL positive (D, upper panel). In non-adherent and unstimulated RPTEC, only very few cells could be observed and they were marginally TUNEL positive (D, lower panel). RPTEC stimulated with TNFα revealed a single detectable non-adherent cell that was TUNEL positive. The cells were counterstained with DAPI to ascertain their presence. As demonstrated by confocal microscopy, FasL did not bind to unstimulated RPTEC, while stimulation with TNFα resulted in strong binding of exogenously added FasL to the cell membranes (E, upper and lower panel, respectively). The FasL-FasR interaction induced apoptosis in RPTEC. Unstimulated adherent cells were TUNEL negative, while Annexin V stained weakly adherent unstimulated RPTEC. In this context, no non-adherent cells could be detected. When stimulating RPTEC with TNFα followed by addition of FasL, both adherent and non-adherent cells were TUNEL-positive (F). Similarly, TNFα-stimulated and FasL-treated adherent and non-adherent RPTEC were strongly Annexin V-positive. The cells were counterstained with DAPI to ascertain their presence. Significances: *P ≤ 0.05; ***P ≤ 0.0005.