2013;502(7470):254C257. present in the primary cilium associated membrane remains to be identified. The complex formed by polycystin 1 (PC1) and polycystin 2 (PC2) functions as a calcium channel at the primary cilium [19, 20]; in this complex PC1 is a mechanosensor [21]. Our previous studies have shown that PC2 is not involved in the autophagy cascade leading to cell size regulation in response to fluid flow [2]. However, it remains a possibility that PC1 is upstream of FLCN to regulate this cascade and cell size in kidney epithelial cells. Further experiments should challenge this hypothesis. In conclusion we show that FLCN localized at PAC-1 the primary cilium regulates autophagy and cell size in kidney epithelial cells in response to shear stress induced by fluid flow. Our work is in line with the fact that autophagy is inhibited in clear cell tumors from a BHD patient [7]. Further studies should address whether this physiological response is altered in BHD patients. MATERIALS AND METHODS Cell culture and siRNA transfection Human kidney HK2 cells (from ATCC) and Birt-Hogg-Dube syndrome associated FLCN-null human kidney UOK 257 cells (as well as FLCN-restored UOK257-2 cells) (from Dr Laura Schmidt (National Cancer Institute, NIH, Bethesda)) were cultured in Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10% FCS at 37C and 5% CO2.For the starvation experiments, cells were cultured in Earle’s balanced salt solution (EBSS) for the indicated times. siRNA transfections were performed using Lipofectamine RNAi Max (Invitrogen, Life Technologies) according to the manufacturer’s instructions. Two siRNA oligomers were used for each target at a final concentration of 20 nM. All siRNAs were purchased from Qiagen and the references are as follows: Control (SI1027281); FLCN (SI05121417 and SI00387660); FNIP1 (a): (SI03222611 and SI05001766). Shear Rabbit Polyclonal to Transglutaminase 2 stress induction HK2 cells were seeded (2.25105 in 150 l of culture medium) into a microslide I0.6 Luer chamber (channel dimensions: 50 x 5 x 0.4 mm, Ibidi) and cultured for 96 h to allow proper polarization and epithelial differentiation. The microslides were connected to a fluid flow system which contains an airpressure pump and a two-way switch valve PAC-1 that pumps PAC-1 the culture medium unidirectionally between two reservoirs through the flow chamber at a rate corresponding to a shear stress of 1 1 dyn/cm2. The control cells (static) were set up in the same microslides Luer chambers and maintained in culture as long as the flow-subjected cells. Protein extraction, immunoblot analysis and antibodies Cells in microslides were washed twice with ice-cold PBS and lysed on ice with 150 l of 1X Laemmli buffer (60 mM Tris-HCl pH=6.8, 2% SDS, 10% Glycerol, bromophenol blue, supplemented with 100 mM DTT) for 30 min. Samples were boiled for 10 min at 95C, separated by SDS/PAGE and then transferred onto Nitrocellulose membranes. Western blot analysis was performed with specific antibodies and the antigenCantibody complexes were visualized by chemiluminescence (Immobilon Western, Merck Millipore). The following antibodies were used in immunoblotting: rabbit-anti LC3 (Sigma, Cat#L7543); rabbit-anti-FLCN (Cell signaling, Cat#3697); rabbit-anti-FNIP1 (Abcam, Cat#ab134969); rabbit-anti-AMPK (Cell signaling, Cat#2532S); rabbit-anti-p-AMPK (T172) (Cell signaling, Cat#2535); mouse-anti-actin (Millipore, Cat#1501); rabbit-anti-ATG16L1 (MBL, Cat#PM040); PAC-1 rabbit-anti-IFT20 (Proteintech, Cat#13615-1-AP); rabbit-anti -catenin (Cell signaling, Cat#8480); rabbit-anti-LKB1 (Cell signaling, Cat#3050); rabbit-anti-S6 ribosomal protein (Cell signaling, Cat#2217); rabbit-anti-p-S6 ribosomal protein (S240/244) (Cell signaling, Cat#2215); rabbit-anti-Tuberin/TSC2 (Cell PAC-1 signaling, Cat#4308); rabbit-anti-p-Tuberin/TSC2 (T1462) (Cell signaling, Cat#3617). Secondary HRP conjugate anti-rabbit IgG (GE Healthcare) and HRP conjugate anti-mouse IgG (Bio-Rad). Immunofluorescence and microscopy Cells were fixed either.