Deletion of Flot-2 enhanced DISC formation and caspase 8 mediated extrinsic apoptosis.

<p>(A) - (B): Beas 2B cells were transfected with Flot-2 siRNA or control siRNA, and exposed to hyperoxia or room air. After 4h, cells were harvested and subjected to Co-IP. (A) Samples were immunoprecipitated with anti-Fas antibodies, followed by Western Blot using anti-FLIP, caspase-8 and Fas antibodies respectively. (B) Samples were first immunoprecipitated with anti-caspase 8 antibodies, followed by Western Blot using anti-FADD antibodies. (C) Over-expression of Flot-2 inhibited DISC formation: Flot-2 was over-expressed in Beas 2B cells. Empty vectors were used as controls. Transfected cells were then exposed to hyperoxia or room air. After hyperoxia (4h), cells were harvested and subjected to Co-IP assays. Samples were first immunoprecipitated with anti-Fas antibodies, followed by Western Blot Analysis using anti-FADD, caspase-8 and Fas antibodies respectively. (D) Deletion of Flot-2 up-regulated the cleaved caspase-8. Beas 2B cells were transfected with Flot-2 siRNA and control siRNA, and exposed to hyperoxia or room air for the indicated time. Both total and cleaved caspase-8 was detected. (E) Deletion of Flot-2 enhanced caspase-8 activation after hyperoxia. Flot-2 silenced Beas 2B cells were exposed to hyperoxia and room air. After 48h, cells were collected and caspase-8 activity was analyzed using flow cytometry and Vybrant® FAM Caspase-8 Assay Kit as described in material and methods. All figures above represented three independent assays with similar results.