Article Figures & Data
Figures
- Figure 1. Generation and characterization of the ovinized SH-SY5Y cell lines.
(A) Generation of SH-SY5YΔPRNP cell lines expressing the ovine VRQ PrPC variant and its subsequent infection with the PG127 strain of sheep-derived prions passaged in tg338 mice. HindIII and EcoRI restriction sites were used to clone the ovine PRNP construct. Ticks in the plasmid map correspond to increments of 1,000 base pairs. hSP, human signal peptide (purple); oSP, ovine signal peptide (blue); hovS, monoclonal ovSH-SY5Y; povS, polyclonal ovSH-SY5Y; ovSH-SY5Y, SH-SY5YΔPRNP transfected with a plasmid harboring the sequence for ovine PRNP (ovPRNP). (B) Western blot analysis comparing the expression levels of PrPC in hovS and povS with those in wt SH-SY5Y and in the human cell lines U251-MG and LN229. HovS and povS showed similar PrPC expression levels as U251-MG and LN229, whereas the levels in wt SH-SY5Y were slightly lower. SH-SY5YΔPRNP cells were used as negative control and actin as loading control. The anti-PrP antibody POM2 was used for detection. (C) Confocal imaging to detect cell surface exposed PrPC on hovS and povS. hovS and a subpopulation of povS showed a strong signal for cell surface exposed PrPC, whereas no detectable signal was visible for SH-SY5YΔPRNP. LN229 cells were used as positive control. The anti-PrP antibody POM1 (here and henceforth) was used for detection of PrP.
Source data are available for this figure.
Source Data for Figure 1[LSA-2020-00814_Sdata1.tif]
- Figure 2. PG127-infected povS and hovS show altered electrophoretic profiles, formation of protease-resistant PrPSc, and enhanced cytopathic effects.
(A) Western blot analysis of PG127-infected povS and hovS cells indicated a different glycosylation pattern, a shift in the electrophoretic mobility, and partially protease-resistant PrPSc, when compared with noninfectious brain homogenate (NBH) exposed–hovS. Rightmost lanes: NBH- and PG127-infected brain homogenate. PG127-infected SH-SY5YΔPRNP lysate was used as negative control. -PK = non–PK-digested. (A, B) Same samples as in (A), but digested with proteinase K (PK). PrPSc is visible in PG127-infected hovS and the PG127 inoculum. No detectable bands are visible for PG127-infected povS, possibly because of the lower expression of PrPC in these cells. (C) Western blot analysis of pellets and supernatants of cell lysates. The aggregate-enriched pellets of PG127-infected povS and hovS displayed a stronger signal for both total PrP and PrPSc. PG127-infected SH-SY5YΔPRNP, NBH, and the original PG127 inoculate were used as controls. (D) Western blot analysis of pronase E–digested PG127-infected hovS, to investigate the presence of PK-sensitive PrPSc. PG127-infected hovS differed again in their protease resistance pattern from those of the original PG127 inoculum. NBH, non-digested PG127 inoculum and lysate of non-digested PG127-infected hovS were used as controls. (E) ELISpot assay of PG127-infected hovS and povS, visualizing cells harboring PrPSc. Membranes were exposed to decadic dilutions of PG127-infected hovS and povS cell suspensions, PK-digested, and stained with POM1. Positive cells were counted on membranes with 400 cells, as higher cell numbers led to signal saturation in hovS cells. Quantification of positive spots (three replicates) revealed 86% ± 12.5% of infected cells for hovS, and 12% ± 2.7% for povS. No positive spots were detected for NBH-treated hovS and povS and PG127-infected SH-SY5YΔPRNP at 40,000 cells. Data in the graph represent the mean ± SD. (F) Phase-contrast image of PG127-infected hovS showing intracellular accumulation of vacuoles (arrows). Scale bar = 50 μm. (G) PG127-infected hovS showed a slower growth rate than NBH-treated hovS over 180 h in culture. Live images were recorded from n = 6 wells for each condition. ***P = 0.0004 (t test at final time point).
Source data are available for this figure.
Source Data for Figure 2[LSA-2020-00814_Sdata2.tif]
- Figure 3. Self-propagating activity and transmissibility of PG127-infected hovS and povS.
(A) The seeding activity of PG127-infected or noninfectious brain homogenate (NBH)–treated hovS, povS, and SH-SY5YΔPRNP lysates was assessed by RT-QuIC (diluted 1:50 and 1:250). PG127-infected hovS, and to a lesser extent, povS cells induced de novo PrP aggregate formation at both dilutions, whereas cell lysates of either NBH-treated or PG127-infected SH-SY5YΔPRNP used as negative controls did not yield a positive signal. Creutzfeldt–Jakob disease (CJD) and non-CJD brain homogenates were used as positive and negative controls for the amplification reaction. Samples were analyzed in quadruplicates. (B) Western blot analysis of serial transmissibility. PG127-infected hovS cell lysates (PG hovS) were used to transmit prion infectivity to fresh hovS cultures. Lysates of undigested and proteinase K–digested hovS exposed to PG127-infected hovS lysates displayed the same electrophoretic profiles as the original lysates. Lysates of hovS exposed to NBH-treated hovS and PG127-infected SH-SY5YΔPRNP cells were used as negative controls. NBH and PG127 inoculum were loaded as additional controls (rightmost lanes). (C) Lysates (20 μl) of PG127-infected hovS and SH-SY5YΔPRNP or NBH-treated hovS were intracerebrally inoculated into tg338 mice. All mice succumbed to disease upon inoculation with PG127-infected hovS lysates with an incubation time of 72 ± 2.5 d. Mice inoculated with control lysates do not show any clinical sign of disease >130 (dpi). n = 6 for each condition. (D) Lysates of PG127-infected hovS (diluted 1:50) were analyzed for propagation efficiency and substrate specificity by PMCA using substrates from various species and of different genotypes (sheep VRQ/VRQ [tg338], sheep ARQ/ARQ [tgARQ], bovine [tgbov], human 129Met [tg650], and human 129V [tg361]). PMCA reactions of the third round were analyzed for PrPSc by Western blotting. Lysates of PG127-infected hovS cells showed positive seeding reactions only with the ovine substrates. NBH-treated hovS and PG127-infected SH-SY5YΔPRNP were used as negative controls and PG127, BSE, and sCJD prions amplified with the respective substrates as positive controls. Reference: PG127 inoculum used to control for signal intensity and band shifts. One representative data set from three experiments is shown.
Source data are available for this figure.
Source Data for Figure 3[LSA-2020-00814_Sdata3.tif]
