Fig 1.
Effects of N-glycan inhibitors NGI-1 and kifunensine on PrPC surface expression in several cell types and PrPSc load in CAD5 cells chronically infected with mouse prions or hamster prions.
(A) Schematic showing N-linked glycan biosynthesis in the ER/Golgi indicating enzymatic processes inhibited by NGI-1 and kifunensine. The lipid-linked oligosaccharide is transferred en bloc to asparagine side chains of nascent polypeptides by the enzyme oligosaccharyltransferase (OST). After transfer to the polypeptide, the glycan undergoes a series of glucose and mannose trimming steps before addition of other residues. Inhibition of discrete steps in this process is enabled using indicated small molecules, NGI-1 and kifunensine. Beauchemin, K. (2025). Adapted from “Protein Glycosylation in the ER.” Retrieved from https://app.biorender.com/biorender-templates (B) Cell surface PrPC levels after treatment with 5 μM NGI-1 or 5 μM kifunensine in culture media for 72 hours, as measured by flow cytometry compared to vehicle-treated controls. Asterisks represent significance values from unpaired t-tests as follows: ***p 0.001, ****p
0.0001. (C) Western blot showing the loss of PK-resistant PrPSc in the lysates from CAD5 cells chronically infected with hamster Hyper or 263K prions. (D) Western blot showing the loss of PK-resistant PrPSc in the lysates from CAD5 cells chronically infected with mouse RML or 22L prions. Cells were treated with 10 μM kifunensine, 5 μM NGI-1, or vehicle controls in the cell culture media for one week. Biological triplicates are shown for each prion strain and treatment condition.
Fig 2.
Effects of NGI-1 supplementation on PrPSc conversion in a cell-free amplification system and siRNA knockdown of targets of NGI-1 on PrPSc levels.
(A) Western blot showing the amplification of PK-resistant PrPSc from brain homogenate PMCA reactions supplemented with DMSO or NGI-1. (B) Representative flow cytometry plot showing effect of siRNA knockdown of Stt3a, Stt3b, or Stt3a and Stt3b combination on surface PrPC levels in WT CAD5 cells. (C) Quantification of flow cytometry PrPC surface expression in undifferentiated CAD5 cells treated with siRNAs targeted at Stt3a, Stt3b, or Stt3a and Stt3b in combination. Points represent individual samples from biological triplicates. Asterisks represent significance values from unpaired t-tests as follows: **p 0.01, ns = not significant. (D) Western blot showing the loss of PK-resistant PrPSc in the lysates from CAD5 cells chronically infected with 22L prions after siRNA knockdown of Stt3a, Stt3b, or Stt3a and Stt3b in combination.
Fig 3.
Effects of NGI-1 treatment on PrPC localization and global structure.
(A) CAD5-PrP-MYC cells were fixed with 4% paraformaldehyde and stained with anti-MYC antibodies to visualize PrP and cholera toxin subunit B (CTB) as a marker for lipid rafts. Single channel and merged confocal images are shown as indicated; scale bar, 10 μm. (B) Quantification of degree of colocalization between PrP puncta and CTB-positive regions. Significance values from unpaired t-tests are represented as follows: ns = not significant. (C) Western blot showing total PrP in supernatant or pellet fractions from CAD5 cells treated for 72 hr with 5 µM NGI-1 or DMSO, lysed, then subjected to centrifugation at 100,000 x g for 1 hr at 4°C in biological triplicate.
Fig 4.
Effect of NGI-1 on amplification of PrPSc seeds in cell lysate PMCA reactions.
(A) Western blot showing cell lysate PMCA reactions containing cell-derived RML seed and cell lysate substrate from DMSO or NGI-1-treated moRK13 cells expressing PrPC. (B) Western blot showing reconstituted PMCA reactions containing cell-derived RML seed, cell lysate substrate from DMSO or NGI-1-treated moRK13 cells that did not express PrPC, and immunopurified PrPC from mouse brain tissue. (C) Western blot showing reconstituted PMCA reactions containing cell-derived RML seed, cell lysate substrate from DMSO or NGI-1-treated moRK13 cells that did not express PrPC, and immunopurified PrPC from DMSO-treated moRK13 cells expressing PrPC.
Fig 5.
Effect of NGI-1 on PrPSc load in non-dividing cells that are chronically infected with mouse or human prions.
(A) Western blot showing the loss of PK-resistant PrPSc in the lysates from non-dividing moPrP RK13 cells chronically infected with 22L prions. Cells were cultured in the presence of 1 µg/mL doxycycline for duration of experiment to induce PrP expression unless otherwise indicated. Cells were treated with 10 µM NGI-1 or DMSO equivalent for one week with no splitting. Biological triplicates are shown for each condition. (B) Log SD50s per mg tissue of RT-QuIC seeding activity from human sCJD-infected cerebral organoids treated therapeutically with 10 µM NGI-1 or DMSO for 30 days or (C) 60 days. Each data point represents the log SD50 from an individual organoid calculated by performing endpoint dilutions, each dilution tested in quadruplicate. Asterisks represent significance values from one-way ANOVA as follows: *p 0.05, **p
0.01, ns = not significant.