Fig. 3: Sam68, FADD, and TRAIL modulate cfDNA release in human non-nontumorigenic MCF-10A cells through apoptotic pathways.

a Immunoblot analysis of Sam68 and FADD after CRISPR-mediated knockout (KO) in the MCF-10A background. TWT = Targeted Wild-Type. b Quantification of DNA release from MCF-10A KO cell lines in culture. Individual cell lines shown, with data representing mean fold change ± SEM internally normalized to cell concentration for each cell line and then normalized to control; n = 3 biologically independent samples. Electropherograms were individually run at least n = 3 times and representative traces were selected. c Quantification of DNA release from Sam68 KO MCF-10A cell lines rescued by Sam68-GFP overexpression. Parental and TWT were grouped and labeled control, and two Sam68 KO3 and KO4 were grouped. Data represent mean fold change ± SEM internally normalized to cell concentration for each cell line and then normalized to control; n = 4 for all lines except n = 3 for Sam68 TWT before combining. Electropherograms were individually run at least n = 3 times and representative traces were selected. d Quantification of DNA release from FADD KO MCF-10A cell lines rescued by FADD-GFP overexpression. Parental and TWT were grouped and labeled control, and two FADD KO cell lines were grouped. Data represent mean fold change ± SEM internally normalized to cell concentration for each cell line and then normalized to control; n = 3 for all GFP-expressing lines and n = 4 for each FADD-GFP expressing line before combining. Electropherograms were individually run at least n = 3 times and representative traces were selected. e Cell growth assay of KO MCF-10A cell lines. Parental and TWT were grouped and labeled control. All four Sam68 KO cell lines and both FADD KO cell lines are respectively grouped. Data represent mean cell concentration ± SEM; n = 3 for each cell line before combining. f Annexin V and Propidium Iodide (PI) assay of KO MCF-10A cell lines. Parental and TWT were grouped and labeled control. All four Sam68 KO cell lines and both FADD KO cell were grouped, respectively. Data represent mean signal (RFU for PI; RLU for Annexin) ± SEM internally normalized to cell concentration for each cell line and then normalized to control; n = 6 biologically independent samples for each cell line before combining. g Cytotoxic assay of MCF-10A KO panel treated with 1 ng/mL TRAIL ligand. Parental and TWT were grouped and labeled control. All four Sam68 mutant cell lines and both FADD mutant cell lines were grouped, respectively. Data represent mean percent survival ± SEM as normalized to vehicle of each cell line condition; n = 3 biologically independent samples for each cell line before combining. h Quantification of DNA release from MCF-10A KO panel treated with 1 ng/mL TRAIL ligand. Parental and TWT were grouped and labeled control. All four Sam68 mutant cell lines and both FADD mutant cell lines were respectively grouped. Data represent mean fold change ± SEM internally normalized to cell concentration for each cell line and then normalized to control; n = 3 biologically independent samples for each cell line before combining. i Quantification of DNA release from MCF-10A KO cell lines treated with 20 μg/mL ZVAD-FM-K. Parental MCF-10As, Sam68 KO3/4 mutant cell lines, and both FADD mutant cell lines were respectively grouped. Data represent mean fold change ± SEM in DNA release internally normalized to cell concentration for each cell line and then normalized to control vehicle; n = 3 biologically independent samples for all untreated lines and FADD KO1, n = 4 for treated lines for each cell line before combining. All statistics were ANOVA with Dunnett’s multiple comparison test at endpoint.