Lung surfactant is stored in lamellar bodies and exocytosed following fusion of the lamellar bodies with the plasma membrane of alveolar type II (AT2) cells [1]. A number of proteins have been shown to be involved in surfactant secretion including SNAREs, NSF, α-SNAP and annexin A2 [2, 3]. Lipid rafts enriched in SNAREs are crucial for surfactant secretion [4]. Flotillins are lipid raft marker proteins. There are two isoforms, flotillin-1 and flotillin-2. Both are expressed in many cell types including AT2 cells [4]. Several functions of flotillins have been reported including interaction with protein kinases and the cytoskeleton; formation of filopodia; insulin signaling; axonal regeneration and cell-cell contacts and membrane trafficking [5, 6]. The evidence for a role of flotillins in exocytosis is lacking except for a recent study which indicated the participation of flotillin-1 in mast cell exocytosis [7].

We investigated the functional role of flotillins in surfactant secretion by decreasing the expression of flotillin-1 and-2 using RNA interference. We used an adenoviral vector to express shRNAs because of its high transduction efficiency in primary cells. This vector contained a shRNA sequence under the control of the mouse U6 promoter with EGFP expression under the control of the CMV promoter (Supplementary information, Materials and Methods). We chose 3 siRNA sequences targeted to different regions for each isoform: 118-137, 522-540 and 584-603 of flotillin-1 [F-1 shRNA (A),(B) and (C)] and 53-73, 602-620 and 1163-1182 of flotillin-2 [F-2 shRNA (A),(B) and (C)]. The construct with irrelevant sequences served as a virus control (VC). The cells which were not transduced with any virus