Fig 2: The first hydrophobic region of Pen-2 is not a TMD. a. Sequence of Pen-2 HP1, SREBP TMD2 and Pen-2-SREBP chimeras. b. Cell membranes from stable cell lines expressing CT11 tagged Pen-2 (lanes 1–3) or Pen-2 TMD1 chimera (lanes 4–6) were treated with proteinase K in the absence or presence of Triton X-100 and resulting products were subject to immunoblotting with PNT2 (upper panel) or CT11 (lower panel) antibodies. c. Cell membranes from Pen-2 chimeras with substitutions of segments from the SREBP TMD2 were treated with proteinase K in the absence or presence of Triton X-100 and resulting products were subject to immunoblotting with PNT2 (upper panel) or CT11 (lower panel) antibodies. Molecular mass markers are shown on the left in kilodaltons. d. Pen-2 model proposed in Refs 17, 18 and 19. E. Model for Pen-2 TMD1. F. Revised Pen-2 model

Fig 3: Rescue of ?-secretase Function in Pen-2-deficient fibroblasts by Pen-2 and the PPS chimera. a. mN?E Processing in transiently cotransfected cells. b. APPswe processing in transiently cotransfected cells

Fig 4: BBR induced AMPK activation was diminished in AXIN1 knockdown and AXIN1-/- cells. BBR’s effect was not influenced on phosphorylation of AMPKa, Raptor and ULK1 in PEN2 knockdown cells (A). BBR’s effect was decreased on stimulating AMPK activity in AXIN1 knockdown cells (B, C). HCT-116 cells were transfected separately with PEN2, AXIN1 and non-sense control (NC) siRNAs. 24 h after transfection, cells were treated with DMSO, BBR (2.5 µM) and metformin (250 µM) for 20 h, respectively. BBR’s effect was inhibited on promoting AMPK activation in AXIN1-/- cells (D, E). AXIN1 was knocked out in HCT-116 cells and the monoclonal transfectants were selected as described in Methods. The wild type and AXIN1-/- cells were starved overnight and treated with DMSO, BBR (2.5 µM) and metformin (250 µM) for 20 h, respectively. The ratio was set as 1 in the NC siRNA transfected (A–C) and wild type (D, E) cells treated with DMSO, respectively. Data are expressed as the mean ± SEM of three separate experiments; * p < 0.05, ** p < 0.01 versus that of the vehicle (DMSO) treated group in the same transfected cells; # p < 0.05, ## p < 0.01 versus that of the corresponding treated group in the NC siRNA transfected or wild type cells.

Fig 5: Schematic diagram illustrates the different mode of action on AMPK activation between BBR and metformin. Metformin activates lysosomal AMPK through PEN2 and AXIN1 after transport into the cellular cytosol (Zhang et al., 2016; Ma et al., 2022). BBR activated lysosomal AMPK through AXIN1 but not PEN2. After interaction with AMPKa, UHRF1 recruits PP2A to dephosphorylate AMPK in the nucleus (Xu et al., 2022). And BBR is reported to bind with UHRF1 (Gu et al., 2020). BBR inhibited the dephosphorylation process of AMPK by reducing the interaction of UHRF1 with AMPKa1 and promoting UHRF1 degradation.