Fig. 5: Apoptosis is the major controller of cfDNA release across nontumorigenic and cancer cell lines. | Communications Biology

Fig. 5: Apoptosis is the major controller of cfDNA release across nontumorigenic and cancer cell lines.

From: An in vitro CRISPR screen of cell-free DNA identifies apoptosis as the primary mediator of cell-free DNA release

Fig. 5

Correlation of Annexin V (a) and Propidium Iodide (b) signal to cfDNA release. Data represent mean DNA release normalized to cell concentration and Annexin V or PI signal (RLU or RLU, respectively) normalized to cell concentration; n = 3 for each cell line, statistics were Pearson correlation with outlier removal using the ROUT method. Out of the 24 cell lines in the panel, 3 were removed in (a) and 2 were removed in (b). c Quantification of DNA release from cancer cell lines upon caspase inhibition with 20 μM ZVAD-FM-K. Data represent mean fold change ± SEM in DNA release normalized to cell concentration for each cell line, then to vehicle; n = 3 biologically independent samples. d Electropherograms were individually run at least n = 3 times and representative traces were selected for samples quantified in (c). e Genes plotted by their β-scores in both cfDNA and gDNA arms of A549 CRISPR screen. Putative hits are highlighted and grouped by shared function (green, apoptotic; yellow, unknown). f Gene ontology of genes from the MCF-10A screen determined as putative hits. Data are -log p-values derived from PANTHER gene ontology, and include enriched pathways in biological process, molecular function, and cellular component. g Immunoblot analysis of BCL-XL after CRISPR-mediated knockout (KO) in the A549 background. TWT = Targeted Wild-Type. h Quantification of DNA release from A549 KO cell lines in culture. Parental cells and TWT cells were averaged and labeled control. All four BCL2L1 KO cell lines were grouped. Data represent mean fold change ± SEM in DNA release normalized to cell concentration for each cell line then normalized to control; n = 4 biologically independent samples for each cell line before combining. Electropherograms were individually run at least n = 3 times and representative traces were selected. i Annexin V and Propidium Iodide (PI) assay on all generated A549 cell lines. Parental cells and TWT cells are combined. All four BCL2L1 KO cell lines were grouped. Data represent mean signal (RFU for PI or RLU for Annexin) ± SEM normalized to cell concentration then overall to Control; n = 6 biologically independent samples for each cell line before combining. All statistical significance shown was derived from student’s t test.

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