Novel and effective plasmid transfection protocols for functional analysis of genetic elements in human cardiac fibroblasts

Figures

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  Fig 1. Chemical transfection of primary human fibroblasts. (A) Schematics of the assay procedure. Human fibroblasts were collected from human tissues and expanded up to passage 8 or 9. Various vectors were transfected for luciferase-based reporter assays and fluorescent protein-based protein localization studies. (B) A GFP-expressing plasmid was transfected into primary human cardio-fibroblasts using various reagents according to the manufacturers’ protocols. A lentiviral vector was infected into fibroblasts as a positive reference. Cells were observed every 24 hours from the transduction until 72 h. After 72 h, propidium iodide (PI) was used to stain dead cells. Scale bar is 500 μm. (C) Fluorescent intensity comparison of GFP and PI in three biological replicates. GFP images taken 24 h after the transfection were used for analysis. PI images were taken at 72 h. The numbers 1–14 correspond to the reagent numbers in Fig 1B. The bar graph for the lentivirus data (positive control) is colored light green and light red in (C) and (D), respectively. Bar graphs represent the means of the biological triplicates, except for PI of R2, which is the mean of duplicate; error bars are the standard…

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  Fig 2. Differences in cell viability by plasmid purification methods. (A-B) Blank plasmid pUC19 was prepared by five commercially available mini- and Midi-kits, CsCl-EtBr centrifugation, and Miraprep protocol. Cell viability (WST-8) and cytotoxicity (LDH) were measured after transduction into cardiac fibroblasts. Bar graphs are the means of the biological duplicates and error bars showing the standard error of the mean. (A) pUC19 is a basic cloning vector, and does not express proteins by gene transfer. pUC19 samples were prepared by 8 different purification methods (M1-M8) and were introduced into HCF with GenomONE-GX (R4) similarly to the data represented in Fig 1. WST-8 were measured 48 hours later. Methods and kits are listed in descending order of expected sample purity, except for the Miraprep samples. As for the purity of Miraprep samples, it depends on the kits applied, so the bar graphs are separated from the other samples. The un-transfected condition means HCF in which neither reagents nor plasmids were added to the medium. (B) Measurement of supernatant LDH of HCF cells 48 hours after gene transfer. The medium was changed once at 24 h after the transfection, and LDH was…

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  Fig 3. Plasmid transfection activates dsDNA receptors and downstream cytokines. (A-B). RT-qPCR analysis of double-stranded DNA receptors (TLR9, AIM2, CGAS, and STING1) and inflammatory cytokines (TNFA, IL6, IL1B, IFNA1, and IFNB1). Blank plasmid pUC19 was transfected into HCF. HCF transfected by only reagent R4 without plasmid acted as negative controls, and its values were set as 1.0. Box plots represent median, 10th, and 90th percentiles from the biological quadruplicates, except for that of TNFA, which is triplicate. Statistical analysis was performed by one-way ANOVA & post-hoc Tukey. Asterisks represent different p-values (*: p < 0.05; **: p <0.01; ***: p < 0.001).

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  Fig 4. Optimization of plasmid transfection protocols. (A) Schedule of optimization experiments. The cells were seeded on day 0 and perform the gene transfer was performed on day 1. Medium replacement was done at 2, 4, 6, 8, or 24 h. At 24 h, the media of all conditions was replaced, and the amount of luciferase secreted into the medium during 24 h from Day 2 to Day 3 was compared between different conditions. CCK-8 assay was performed on Day 3. (B) Luciferase activity after transfection into HCF cells with Reagent #4 (R4) under 100 unique conditions. The variable parameters in R4 are the addition of KALA (cationic amphiphatic) peptide or not, the addition of an inhibitor of innate immunity or not, and the incubation time from mixing reagent/plasmid to applying to the cells, and the timing of first medium change. Each condition was performed twice, and one square corresponds to one condition, divided by a diagonal line to show the repeated results. The luciferase activity is divided into top 1%, 5%, 10%, 25%, and 50% percentile, with the higher activity indicated by a darker color. (C) Luciferase activity after transfection into HCF cells with Reagent #8 under 50 conditions; the…

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  Fig 5. Promoter assay with optimized protocol for primary human cardiac fibroblasts. (A) Map of the plasmids used in the ubiquitous promoter assay. The secreted luciferase GLuc and the green fluorescent protein DasherGFP linked by IRES sequences are expressed downstream of the indicated promoters. In addition, WPRE, a post-transcriptional regulatory element, and a synthetic polyA signal are inserted downstream of the GFP for stabilization of the expressed mRNA. (B) Comparison of the viral and synthetic promoters’ activities in HCF. Each plasmid with different promoters was transfected into HCF. GLuc activity in the medium was measured 48 hours after the transduction. All experiments were performed in quadruplicates, bars represent means and error bars are standard error of the mean. (C) Candidate isolation of predicted endogenous enhancer-promoter sequences. The dark blue horizontal bar represents exons and introns of human Vim mRNA (NM_003380.5). The histogram represents the basewise conservation of 100 vertebrates analyzed by PhyloP. In ENCODE Candidate Cis-Regulatory Elements (cCREs), Red boxes indicate promoter-like signature and orange boxes proximal enhancer-like signature. All…