Journal: Applied Microbiology and Biotechnology

Article Title: Targeted knockdown of ATM, ATR, and PDEδ increases Gag HIV-1 VLP production in HEK293 cells

doi: 10.1007/s00253-024-13389-8

Figure Lengend Snippet: Growth kinetics and VLP production of HEK293 cells transfected with Gag::eGFP plasmid carrying the shRNAs targeting DNA-PKcs, ATR, ATM, and PDEδ. a. Viable cell density (solid lines) and viability (dotted lines) of the different conditions along the studied time course ( n = 3). b. Transfection efficiency measured as percentage of GFP-positive cells by flow cytometry. c. VLP production of the different studied conditions measured via fluorometry as relative fluorescence units (RFU) in the supernatant at 72 hpt. d. VLP titers in the supernatant measured by flow virometry. e. Particle size distribution in the VLP-containing supernatants measured by nanoparticle-tracking analysis at 72 hpt ( n = 3). VLP titers are indicated over the peaks. f. Pixel density analysis of western blot of the relative expression of ATM, ATR, DNA-PKcs and PDEδ proteins compared to CC and normalized using β-actin as housekeeping protein. In all panels: blue is for control transfected without any shRNA (CC), light grey for condition transfected with plasmid carrying a scramble shRNA (SC), brown for shRNA against DNA-PKcs (DNA), dark grey for shRNA against ATR (ATR), pearl for shRNA against PDEδ (PDE) and cyan for shRNA against ATM (ATM). Error bars indicate the standard deviation. Statistical significance studied using one-way ANOVA and Dunnett test analysis of the VLPs in the supernatant at 72 hpt of the knockdowns relative to the control condition (’****’ = p -value < 0.0001, ‘***’ = p -value = 0.0003)

Article Snippet: The cell line used in this work is a serum-free suspension-adapted HEK293 cell line (HEK293SF-3F6) kindly provided by Dr. Amine Kamen from the National Research Council of Canada (NRC, Montreal, Canada).

Techniques: Transfection, Plasmid Preparation, Flow Cytometry, Fluorescence, Western Blot, Expressing, Control, shRNA, Standard Deviation

Journal: Applied Microbiology and Biotechnology

Article Title: Targeted knockdown of ATM, ATR, and PDEδ increases Gag HIV-1 VLP production in HEK293 cells

doi: 10.1007/s00253-024-13389-8

Figure Lengend Snippet: Analysis of key production parameters of HEK293 producing VLPs under transient transfection and target-protein knockdowns. a. Budding efficiency of the different conditions measured as the percentage of RFU in the supernatant relative to the total RFU produced. p -values show the statistical significance of the percentage of RFU in the supernatant. b. Intracellular RFU content of the different conditions. c. Regression analysis of the specific productivity (VLPs/cell × day) measured through fluorimetry (dots and green line), flow virometry (inverted triangles and pink line), and NTA (squares and black line) relative to the mean particle size (nm). The points follow the same color legend as in the other panels: navy blue for CC, brown for DNA, grey for ATR, beige for PDE and cyan for ATM. d. Extracellular vesicles (EVs) concentration of the different conditions. e. Percentage of VLPs over the total secreted particles. VLP were measured as fluorescent particles and total particles were measured by dispersion analysis using NTA. In all panels, measurements were carried out using 72 hpt samples. Error bars indicate the standard deviation. Statistical significance was studied using one-way ANOVA and Dunnett test analysis of the knockdowns relative to the control condition (’****’ = p -value < 0.0001, ‘**’ = p -value = 0.0034, ‘*’ = p -value = 0.0118)

Article Snippet: The cell line used in this work is a serum-free suspension-adapted HEK293 cell line (HEK293SF-3F6) kindly provided by Dr. Amine Kamen from the National Research Council of Canada (NRC, Montreal, Canada).

Techniques: Transfection, Produced, Concentration Assay, Dispersion, Standard Deviation, Control