Journal: PLoS Pathogens

Article Title: Infectious Prions Accumulate to High Levels in Non Proliferative C2C12 Myotubes

doi: 10.1371/journal.ppat.1003755

Figure Lengend Snippet: (A) Morphology of C2C12 myoblasts and myotubes (B) Western blot comparison of PrP C expression from mouse brain homogenate, C2C12 myoblasts and myotubes and N2a neuroblastoma cells. Relative loads are based on protein concentration of the brain homogenate (1.78 µg, 0.68 µg protein) and cell lysates (3.4 µg C2C12 myoblasts, myotubes and N2a). Anti-PrP antibody SAF 83 was used.

Article Snippet: C2C12 (CRL-1722) myoblast cells were purchased from the American Type Culture Collection, expanded and aliquots stored in liquid nitrogen and expanded as needed.

Techniques: Western Blot, Comparison, Expressing, Protein Concentration

Journal: PLoS Pathogens

Article Title: Infectious Prions Accumulate to High Levels in Non Proliferative C2C12 Myotubes

doi: 10.1371/journal.ppat.1003755

Figure Lengend Snippet: (A) C2C12 myotubes infected with RML were collected at 4 or 15 days post infection by scraping in PBS, centrifuging and resuspending in 500 µL of water. 30 µL of this cell material was inoculated intracranially into weanling C57Bl/6 mice. A statistically significant difference in incubation period was observed between 4 and 15 days indicative of prion replication p<0.05. Parallel inoculation of serial 10-fold dilutions of RML brain homogenates were performed. (B) Plot of infectivity in each inocula standardized per gram of protein inoculated. C2C12 myotubes infected with RML were collected at 4, 6, 8, 10, 13 and 15 days post-infection as in (A) and bioassayed. Infectivity of the C2C12 derived samples was calculated by comparing the observed incubation periods from each sample with those from brain homogenate dilutions and normalizing the data to protein content. Prion infectivity increases in C2C12 myotube cultures from day 4 to day 15 post-infection.

Article Snippet: C2C12 (CRL-1722) myoblast cells were purchased from the American Type Culture Collection, expanded and aliquots stored in liquid nitrogen and expanded as needed.

Techniques: Infection, Incubation, Derivative Assay

Journal: PLoS Pathogens

Article Title: Infectious Prions Accumulate to High Levels in Non Proliferative C2C12 Myotubes

doi: 10.1371/journal.ppat.1003755

Figure Lengend Snippet: Gene expression profiling was performed from 3 replicate samples of uninfected and infected C2C12 myotube preparations. (A) Confirmation of PrP Sc in cells used for gene expression profiling by immunoblotting with 3F10. Cell lysates were digested with 50 ug/mL of proteinase K (PK50) and 10 µg of protein was loaded in each lane. (B) Gene expression of infected vs. un-infected control cell cultures (N = 3). A scatter plot was created comparing the expression of genes in uninfected cells with that of infected cells. Each data point plotted is the average normalized signal intensity for each gene's expression (uninfected or infected). Data points outside the green lines have greater than 2-fold changes in gene expression. Transcription was globally unchanged in C2C12 cells replicating PrP Sc .

Article Snippet: C2C12 (CRL-1722) myoblast cells were purchased from the American Type Culture Collection, expanded and aliquots stored in liquid nitrogen and expanded as needed.

Techniques: Gene Expression, Infection, Western Blot, Control, Expressing

Journal: PLoS Pathogens

Article Title: Infectious Prions Accumulate to High Levels in Non Proliferative C2C12 Myotubes

doi: 10.1371/journal.ppat.1003755

Figure Lengend Snippet: (A) Time dependent increase in PrP Sc in C2C12 cells. 10 µg of protein (PK−) or protein equivalent (PK+) was loaded in each lane. (B) Relative abundance of PrP Sc in clinically affected mouse brain homogenate and lysates from cell lines (C2C12 myotubes, N2a neuroblastoma cells and SMB scrapie mouse brain cells). All samples are treated with PK (50 µg/mL, final concentration). Relative loading is equivalent to protein amounts prior to proteinase K digestion 0.75 µg, 7.5 µg and 75 µg respectively. Antibody SAF 83 was used.

Article Snippet: C2C12 (CRL-1722) myoblast cells were purchased from the American Type Culture Collection, expanded and aliquots stored in liquid nitrogen and expanded as needed.

Techniques: Concentration Assay

Journal: PLoS Pathogens

Article Title: Infectious Prions Accumulate to High Levels in Non Proliferative C2C12 Myotubes

doi: 10.1371/journal.ppat.1003755

Figure Lengend Snippet: C2C12 cells were infected with 100 µL of 10% brain homogenates. Two days post infection, pentosan polysulfate (PPS) (1 µg/ml final concentration) was added to the media. PPS was kept on the cells until harvested at the designated dates. Antibody SAF 83 was used for western blot detection.

Article Snippet: C2C12 (CRL-1722) myoblast cells were purchased from the American Type Culture Collection, expanded and aliquots stored in liquid nitrogen and expanded as needed.

Techniques: Infection, Concentration Assay, Western Blot

Journal: PLoS Pathogens

Article Title: Infectious Prions Accumulate to High Levels in Non Proliferative C2C12 Myotubes

doi: 10.1371/journal.ppat.1003755

Figure Lengend Snippet: (A) Brain homogenates (BH) from mice clinically affected with prion disease. 10 ul of 1% BH treated with or without 50 µg/ml PK was loaded in each lane. 22L and ME7 prions were generated in tga20 mice. RML prions were from C57Bl/6 mice. PrP Sc was detected with 3F10 antibody. (B) C2C12 cells were infected with brain homogenates from 22L, ME7 and RML. At 4, 8 and 14 days post exposure, cells were lysed, lysates treated with 50 ug/mL of proteinase K and immunoblotted to detect the presence of PrP Sc . 30 µg of protein equivalent was loaded into each lane for 22L and ME7 samples. 10 µl of 0.1% BH was loaded as a control. 10 µg of protein equivalent from RML infected myotubes was loaded.

Article Snippet: C2C12 (CRL-1722) myoblast cells were purchased from the American Type Culture Collection, expanded and aliquots stored in liquid nitrogen and expanded as needed.

Techniques: Generated, Infection, Control

Journal: PLoS Pathogens

Article Title: Infectious Prions Accumulate to High Levels in Non Proliferative C2C12 Myotubes

doi: 10.1371/journal.ppat.1003755

Figure Lengend Snippet: 15 µg of protein was loaded in each lane. RML and HY brain homogenates were 15 µl of 0.1% (A) C2C12 myotubes infected with RML accumulate PrP Sc as detected by the SAF83 monoclonal antibody. SAF 83 recognizes mouse and hamster PrP. (B, C) C2C12 myotubes infected with hamster HY prions probed with SAF83 (B) and 3F4 (C) antibodies. 3F4 recognizes hamster PrP but not mouse, including some HY that persists in culture until day five. SAF83 also recognizes mouse prions, but no signal is detected at day 5. This is likely due to the higher affinity of 3F4 vs. the SAF83 antibody.

Article Snippet: C2C12 (CRL-1722) myoblast cells were purchased from the American Type Culture Collection, expanded and aliquots stored in liquid nitrogen and expanded as needed.

Techniques: Infection