Cell lines and animals

Cells were maintained at 37°C with 5% CO₂ in air in Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS). N2aC24, N2aC24L1-3, and cured N2aC24L1-3 cells were previously established elsewhere [26]. N2aC24 cells were cloned from mouse neuroblastoma N2a cells overexpressing exogenous mouse PrP^C. N2aC24L1-3 cells were cloned from N2aC24 cells persistently infected with 22L scrapie prions. Cured N2aC24L1-3 cells are cured from prion infection by treatment with SAF32 anti-PrP Ab and then maintained in antibody-free DMEM with 10% FBS. ScN2a cells (kindly gifted from Prof Doh-ura, Tohoku University) were N2a cells persistently infected with RML scrapie prions.

Sortilin-KO cells, ΔSort#1 and #2 cells, were established using the CRISPR-Cas genome editing system. N2aC24 cells were transduced with pRGEN-Cas9-CMV (Takara bio, Shiga, Japan) and pRGEN_Mouse-Sort1_U6_SG_1 targeting the sequence (5’-cctgccgccgtcggccaggaccg-3’) (Takara bio), and subjected to limiting dilution cloning. Knockout of sortilin in ΔSort#1 and #2 cells was confirmed by Western blotting.

PrP-KO N2a cells, termed N2aΔPrP cells, were also established using the CRISPR-Cas genome editing system. N2a cells were transduced with pRGEN-Cas9-CMV (Takara bio) and pRGEN_PrP_U6_SG_1 targeting the sequence (5’-accggtggaagccggtatcccgg-3’) (Takara bio), and subjected to limiting dilution cloning. Knockout of PrP^C in N2aΔPrP cells was confirmed by Western blotting. To clone N2aΔPrP cells expressing full-length wild-type PrP^C or PrPΔ23–88, pEF1-moPrP(3F4) and pEF1-moPrP(3F4)Δ23–88 were linearized by Sca I and transfected into N2aΔPrP cells. The cells were treated with G418 and the G418-resistant cells were cloned by limiting dilution, resulting in establishment WT#1 and #2 cells and Δ23–88#1 and #2 cells.

Sortilin-KO (Sort1^-/-) mice used in this study were previously produced elsewhere [11,22]. Sort1^-/- mice having been backcrossed for 10 generations into C57BL/6 were intercrossed with C57BL/6 mice (Charles River Laboratories, Kanagawa, Japan), and the resulting heterozygous mice (Sort^-/+) were then intercrossed to obtain Sort^-/- and wild-type (Sort^+/+) mice.