Fig 2: Chronically Me7 infected PME2 cells deposit PrPd aggregates and differ in their PK sensitivity from RML-infected cells. (A) Laser-scanning microscopy images of uninfected and chronically Me7-infected PME2 subclones. Eleven passages after infection of PME2-1H10 cells with a 10-5 dilution of Me7 brain homogenate, cells were plated out in chamber slides and cultured for 6 days. Cells were treated post fixation according to Materials and Methods and labeled with 5B2 and CellMask Deep Red plasma membrane (CMPM) stain (Thermo Scientific) according to the manufacturer’s specification. Magnifications of infected PME2-1H10 cells are shown in the panel below as indicated. (B) Western blot of brain homogenates CD1 (uninfected), Me7 and RML and cell-adapted prion strains PME2 [Me7] and PME2 [RML] with and without Proteinase K (PK) digestion. (C,D) Pools of three Me7- and RML-infected PME-6D8 clones were ribolyzed and protein concentrations were determined by bicinchoninic acid (BCA). Aliquots of 50 µg cell homogenate were digested with Proteinase K (PK) at the concentration range specified. Representative Western blot are shown in C and PK-sensitivity of PrPSc quantified (D) as specified in Materials and Methods.

Fig 3: Progressive enrichment of Me7-susceptible cells by single cell cloning. (A) Schematic representation of a serial subcloning strategy to identify Me7-susceptible cell clones. Plates of single PK1 cell clones were infected with Me7, while duplicate plates remained uninfected (not shown). The susceptible clone PME2 was expanded at limiting dilution to isolate 2nd generation clones (PME2 progeny). This protocol was repeated to isolate 3rd generation (PME2-6D8) clones. (B) Progressive enrichment of Me7-susceptible cell clones. Data represents the percentage of Me7-positive cell clones in three subsequent subcloning experiments according to A. The total number of clones characterized in experiments 1, 2 and 3 were 720, 960, and 468, respectively. (C) The rate of prion propagation in prion-infected cells increases progressively upon sequential subcloning. Data represents average rates of prion propagation for five representative cell clones and is expressed as number of PrPSc-positive cells per day ± SD. Statistical significance was calculated using Student’s t-test (** p < 0.001) (D) Enrichment of highly Me7-susceptible cells in 3rd generation (PME2-6D8) subclones. Data represents spot numbers of individual clones from two cell passages (passage 3 and 5). An increase in spot number is indicative of perpetuating prion replication. Correlation coefficients R2 for linear regression analysis is shown. (E) Distribution of Me7-susceptible PME2 and PME2-6D8 clones. Cell clones were challenged with a 10-5 dilution of Me7 brain homogenate (10% w/v). After five cell passages, the number of spots was determined by Scrapie Cell assay.