Fig 2: Serine mutagenesis pinpoints Omega signal localization. (A) The amino acid sequence comprising peptide fragment AQP6187–213 is shown. (B) When this peptide fragment was substituted with 5 serine substitutions and C-terminally attached to AGR, this construct fails to localize to the PM of HEK293 cells. In contrast, (C–E) amino acid serine substitutions within the indicated peptide fragments and then C-terminally attached to AGR, these constructs localize in the PM of HEK293 cells (white arrows). Cells were observed microscopically using an upright Olympus BX51WI equipped with 100 × 1 NA objective and images were captured using a Grasshopper3 CMOS camera (FLIR, Richmond, BC, Canada) controlled by Leica LAS X version 3.5 software (available at: https://www.leica-microsystems.com/products/microscope-software/p/leica-las-x-ls/). All figures were created using Adobe Illustrator CS6 (Available at: https://adobe.com/products/illustrator), under an Adobe Inc., Creative Cloud Desktop 2019 shared device license to Case Western Reserve University (CWRU) that operates until 3/31/2022. Scale bars: 8 µm.

Fig 3: AQP6 expressed in transient heterologous transfection constructs rarely reaches the plasma membrane. (A) The N-terminal tGFP-labeled AQP6 construct is shown (tGFP-AQP6). The peptide linker is shown in blue and the C-terminal 10× Histag in black. (B) The C-terminal GFP-labeled AQP6 construct is shown (AQP6-tGFP). The peptide linker is shown in blue and the C-terminal 10× Histag in black. (C) Transient transfection of an AQP6-tGFP construct in HEK293 cells yields very poor to no expression. (D) Transient transfection of tGFP-AQP6 construct in HEK293 cells improves expression but most of the expressed protein is retained in the ER. Scale bars (C, D): 5 µm. (E) Very rarely, the tGFP-AQP6 construct localizes to the PM (white arrows) in HEK293 cells. Scale bar: 3 µm. Cells were observed microscopically using an upright Olympus BX51WI equipped with 100 × 1 NA objective, and images were captured using a Grasshopper3 CMOS camera (FLIR, Richmond, BC, Canada) controlled by Leica LAS X version 3.5 software (available at: https://www.leica-microsystems.com/products/microscope-software/p/leica-las-x-ls/). All figures were created using Adobe Illustrator CS6 (available at: https://adobe.com/products/illustrator), under an Adobe Inc., Creative Cloud Desktop 2019 shared device license to Case Western Reserve University (CWRU) that operates until 3/31/2022.

Fig 4: AGR scanning of the second half of AQP6. (A) Approaching the second half of AQP6 in a similar manner to Fig. 2, peptide residues AQP6158–167 when C-terminally attached to AGR results in a construct that reaches the PM (white arrows). (B) However, when residues AQP6187–213 are C-terminally attached to AGR, the construct does not reach the PM. (C) Although, AQP6232–276 residues (compromising AQP6 C-terminal) attached to AGR does localize to the PM. (D) Interestingly, when a scrambled version of peptide residues AQP6187–213 are substituted for the native AQP187–213 and C-terminally attached to AGR, this construct is able to reach the PM. Cells were observed microscopically using an upright Olympus BX51WI equipped with 100 × 1 NA objective and images were captured using a Grasshopper3 CMOS camera (FLIR, Richmond, BC, Canada) controlled by Leica LAS X version 3.5 software (available at: https://www.leica-microsystems.com/products/microscope-software/p/leica-las-x-ls/). All figures were created using Adobe Illustrator CS6 (Available at: https://adobe.com/products/illustrator), under an Adobe Inc., Creative Cloud Desktop 2019 shared device license to Case Western Reserve University (CWRU) that operates until 3/31/2022. Scale bars: 8 µm.

Fig 5: SM875 lowers the amount of PrP at a post-translational level in different cell lines.Cells were exposed to different concentrations of SM875 or vehicle (0.1% DMSO) for 48 h, lysed, and analyzed by western blotting. a In ZR-75 cells, SM875 suppresses PrP, but not Thy-1, in a concentration-dependent fashion. b Similar effects were observed in cultured L929 fibroblasts. c In N2a cells, SM875 shows a dose-dependent lowering effect of PrP at 1–10 µM. However, in contrast to the other cell lines, the compound showed no effect at 30 µM. All signals were detected by using a specific anti-PrP (D18) or anti-Thy-1 primary antibodies. Red arrowheads indicate the expected sizes of mature, fully glycosylated forms of PrP or Thy-1. Western blotting analysis (i) and graphs reporting the densitometric quantification of signals (ii) are shown. Each signal was normalized on the corresponding total protein lane (detected by UV of stain-free gels) and expressed as the percentage of vehicle (Vhc)-treated controls (*p < 0.05, **p < 0.01, ***p < 0.005, by one-way ANOVA test). d Graphs show the levels of PrP mRNA upon treatment with SM875, as evaluated by RT-PCR. Specific forward and reverse primers were used to amplify endogenous or exogenous, mouse or human PrP transcripts (see Materials and Methods). Relative quantification was normalized to mouse or human HPRT (hypoxanthine–guanine phosphoribosyltransferase). Statistical analyses refer to the comparison with vehicle controls (**p < 0.01, ***p < 0.005, by one-way ANOVA test). Dots represent biologically independent replicates. e HEK293 stably expressing a PrP form tagged with a monomerized EGFP molecule at its N-terminus (EGFP-PrP) were incubated with vehicle (0.1% DMSO) control (i) or SM875 at different concentrations (ii–vi) for 24 h. Fluorescence of the EGFP protein was then visualized with an Olympus BX51WI microscope equipped with reflected fluorescence. Scale bar 50 µm.