human brain tumor cell line u 87 mg  (ATCC)

Journal: bioRxiv

Article Title: Synthetic Genome Shuffling of Poxviruses through Yeast for Next-Generation Oncolytic Platforms

doi: 10.64898/2026.03.06.710085

Figure Lengend Snippet: (A) PCR analysis of the three VACV genomic fragments transformed into yeast, performed on pre-selected TAR-derived clones. (B) Restriction enzyme analysis of plasmid DNA from clones C1 and C2 following transformation into E. coli and plasmid purification. (C) Brightfield microscopy images of plaque assays on A549 cells infected with MVA, VACV, the rescue material from cells transfected with pYCC3-VACV_C1 (clone C1), the rescue material from cells transfected with pYCC3-VACV_C2 (clone C2), as well as rescue samples from MVA-infected cells transfected with pYCC3-VACV_C1 (MVA + pYCC3-VACV_C1) or pYCC3-VACV_C2 (MVA + pYCC3-VACV_C2). A549 cells were seeded at 5 × 10⁵ cells per well in 6-well plates and infected at a multiplicity of infection (MOI) of 10⁻⁵. Images were acquired 48 h post-infection at 4× magnification using brightfield microscopy. (D) Distribution of parental viral genomes within isolated clones obtained from rescue experiments in MVA-infected cells transfected with pYCC3-VACV_C1 or pYCC3-VACV_C2, based on deep sequencing analysis.

Article Snippet: Human tumor cell lines A549 (ATCC CCL-185), HeLa (ATCC CCL-2), MIA-PaCa-2 (ATCC CRL-1420), HCT116 (ATCC CCL-247), and the African green monkey kidney cell line Vero (ATCC CCL-81) were maintained at 37 °C in a humidified atmosphere of 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM, Thermo Fisher Scientific, USA) or McCoy’s 5A medium (LGC Standards, UK), supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, USA) and 100 μg·mL−1 gentamycin (Sigma-Aldrich, USA).

Techniques: Transformation Assay, Derivative Assay, Clone Assay, Plasmid Preparation, Purification, Microscopy, Infection, Transfection, Isolation, Sequencing

Journal: bioRxiv

Article Title: Synthetic Genome Shuffling of Poxviruses through Yeast for Next-Generation Oncolytic Platforms

doi: 10.64898/2026.03.06.710085

Figure Lengend Snippet: (A) Brightfield microscopy images of plaque assays performed on HeLa cells infected with parental viruses (VACV, CPXV, RPXV) and with chimeric viruses rescued from TAR-shuffling clones (cPOX01-02, cPOX02-03, cPOX04-12, cPOX05-14, and cPOX06-18). HeLa cells were seeded at 5 × 10⁵ cells per well in 6-well plates and infected at a multiplicity of infection (MOI) of 10⁻⁵. Images were acquired 48 h post-infection at 4× magnification. (B) Representative comet-shaped plaques observed in A549 cells. A549 monolayers were infected with the indicated viruses, incubated for 48 h, and stained with crystal violet to visualize comet formation, reflecting enhanced viral spread

Article Snippet: Human tumor cell lines A549 (ATCC CCL-185), HeLa (ATCC CCL-2), MIA-PaCa-2 (ATCC CRL-1420), HCT116 (ATCC CCL-247), and the African green monkey kidney cell line Vero (ATCC CCL-81) were maintained at 37 °C in a humidified atmosphere of 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM, Thermo Fisher Scientific, USA) or McCoy’s 5A medium (LGC Standards, UK), supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, USA) and 100 μg·mL−1 gentamycin (Sigma-Aldrich, USA).

Techniques: Microscopy, Infection, Clone Assay, Incubation, Staining

Journal: bioRxiv

Article Title: Synthetic Genome Shuffling of Poxviruses through Yeast for Next-Generation Oncolytic Platforms

doi: 10.64898/2026.03.06.710085

Figure Lengend Snippet: (A–D) A549 (A), HCT 116 (B), HeLa (C), and MIA PaCa-2 (D) cells were infected with serial dilutions of each virus. Cell viability was assessed 4 days post-infection using the CellTiter-Blue assay and normalized to uninfected controls. Data represent the mean ± SD from three independent experiments. (E) EC₅₀ values for each virus in each cell line were calculated by fitting a sigmoidal dose–response curve to the viability data shown in panels A–D. Statistical significance was determined relative to the VACV group using one-way ANOVA: p < 0.05 (*).

Article Snippet: Human tumor cell lines A549 (ATCC CCL-185), HeLa (ATCC CCL-2), MIA-PaCa-2 (ATCC CRL-1420), HCT116 (ATCC CCL-247), and the African green monkey kidney cell line Vero (ATCC CCL-81) were maintained at 37 °C in a humidified atmosphere of 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM, Thermo Fisher Scientific, USA) or McCoy’s 5A medium (LGC Standards, UK), supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, USA) and 100 μg·mL−1 gentamycin (Sigma-Aldrich, USA).

Techniques: Infection, Virus, CtB Assay

Journal: bioRxiv

Article Title: Synthetic Genome Shuffling of Poxviruses through Yeast for Next-Generation Oncolytic Platforms

doi: 10.64898/2026.03.06.710085

Figure Lengend Snippet: (A) Viral amplification in A549 cells. Monolayers were infected at a MOI of 10⁻³. At 3 days post-infection, both supernatants and cells were collected, and viral progeny were quantified by plaque assay. Results are expressed as fold amplification, calculated as the output/input ratio. Data represent mean ± SD from three independent experiments. Statistical significance was assessed against the VACV group using one-way ANOVA: not significant (ns), p < 0.05 (*), p < 0.01 (**). (B) Ratio of EEV to total progeny virus (IMV + EEV). A549 cell monolayers were infected with the indicated viruses at a MOI of 0.1. Viral titers were determined for EEV (supernatant only) and total progeny virus (IMV + EEV; combined supernatants and cells). Data represent mean ± SD from three independent experiments. Statistical significance was assessed against the VACV group using one-way ANOVA: not significant (ns), p < 0.05 (*), p < 0.01 (**).

Article Snippet: Human tumor cell lines A549 (ATCC CCL-185), HeLa (ATCC CCL-2), MIA-PaCa-2 (ATCC CRL-1420), HCT116 (ATCC CCL-247), and the African green monkey kidney cell line Vero (ATCC CCL-81) were maintained at 37 °C in a humidified atmosphere of 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM, Thermo Fisher Scientific, USA) or McCoy’s 5A medium (LGC Standards, UK), supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, USA) and 100 μg·mL−1 gentamycin (Sigma-Aldrich, USA).

Techniques: Amplification, Infection, Plaque Assay, Virus