Fig 2: Effects of different treatments on the AP-5/SPG11/SPG15 complex versus mTORC1. (A) IF labeling of SPG15-GFP (amplified with anti-GFP) in cells that had been treated with siRNA to knock down RagA, the RagC/D GAP FLCN, or the RagA/B GATOR1 GAP subunit NPRL2. To better visualize the labeling and any effects of siRNA depletion, all cells were starved (1 h). These knockdowns have a slight effect on SPG15-GFP recruitment, but they are not as dramatic as a RagC or Ragulator knockdown. Scale bar: 20 µm. (B) Western blots of whole-cell lysates from SPG15-GFP cells treated with siRNA as indicated, with mTOR as a loading control. The blot shows mTOR labeling in a control versus FLCN knockdown, but it is representative of the loading for both knockdowns. The blots are representative of at least two independent experiments. (C) IF labeling of SPG15-GFP (amplified with anti-GFP) and mTOR in cells treated in various ways, showing an inverse relationship between the two. Starvation (1 h) increases SPG15 recruitment while preventing mTOR recruitment, while the drugs rapamycin and Torin1 lock mTOR on the membrane and prevent recruitment of SPG15. Scale bar: 20 µm. (D) IF labeling of SPG11-GFP (amplified with anti-GFP), showing that, like SPG15-GFP, its membrane association is inhibited by rapamycin and Torin1. Scale bar: 20 µm. kd, knockdown.

Fig 3: FIGURE 6: Shear-stress dependent recruitment of ATG16L1 to primary cilium is impaired in FLCN knockdown cells.HK2 cells were transfected with control siRNA (siCTRL) or with siRNA targeting FLCN (siFLCN). 72 h later, they were subjected to fluid flow for 4 days (shear 4D) or not (static 4D). (A) ATG16L1 and actin levels were analyzed by western blot. (B) Cells prone to fluid flow were fixed with methanol, labeled with DAPI, immunostained for ATG16L1 and ARL13B and then analyzed by fluorescence microscopy. Arrowhead indicates presence of ATG16L1 at the basal body. (C) ATG16L1 positive structures at basal body were quantified from experiments shown in (B). Scale bar = 10μm.

Fig 4: HEK293T cells expressing FLCN WT or Mut fused to GFP using the TALEN technology were subjected to immunofluorescent imaging. WT FLCN localizes to both cytoplasm and nucleus, the mutant protein is primarily found in the cytoplasm. Interestingly treatment with MG-132 did not only stabilize the mutant protein which is reflected by a shorter exposure time during image acquisition but also led to a nuclear shift making its localization more similar to the WT protein (bar = 20 μm, exposure time of the GFP and DAPI channel are depicted in millisecond in the upper right corner of each panel)

Fig 5: Insulin‐dependent FLCN phosphorylation at Ser62 by AKT Insulin‐regulated lysosomal localization of mTORC1. A) HEK293T cells were overexpressed Flag‐FLCN, immunoblotting with AKT‐substrate antibody to detect the phosphorylation of Flag‐FLCN. B) HEK293T cells were starved of amino acids and serum for 24 h and then supplemented with insulin for 0, 5, 10, 15, or 30 min, the phosphorylation of Flag‐FLCN was detected by AKT‐substrate antibody. C) Overexpression of Flag‐FLCN in HEK293T cells, and treatment of cells with different types of inhibitors, the phosphorylation of Flag‐FLCN was detected by AKT‐substrate antibody. D) A schematic showing the evolutionarily conserved putative AKT phosphorylation sites, Ser 62 within FLCN. E) Overexpression of the Flag‐FLCN, Flag‐FLCN S62A, or Flag‐FLCN S62D in HEK293T cells, the phosphorylation of Flag‐FLCN was detected by AKT‐substrate antibody. F) Overexpression of different concentrations of HA‐AKT1 or combined with MK2206 in HEK293T cells, the phosphorylation of Flag‐FLCN was detected by AKT‐substrate antibody. G) HEK293T cells were starved of amino acids and serum for 24 h and then supplemented with insulin for 0, 5, 10, or 15 min. The interaction of Flag‐FLCN and HA‐AKT1 were analyzed via a co‐IP assay. H) HEK293T cells were starved of amino acids and serum for 24 h and then supplemented with insulin for 30 min alone or in the presence of MK2206. The interaction of endogenous AKT1 and FLCN were analyzed via a co‐IP assay. I) Immunoprecipitates prepared from cells lysates were used in pulldown assays. In vitro pulldown assay indicated that HA‐AKT1 preferentially interacts with the Flag‐FLCN. J) Immunoprecipitates prepared from lysates cells were used in kinase assays. Immunoblotting with AKT‐substrate to detect the phosphorylation of Flag‐FLCN.