Extended Data Fig. 2: APAF1 and NCF1 regulate EV-DNA packaging.
(a-b) EVs were isolated from control and APAF1 KO cells. (a) Mode diameter of EVs by nanoparticle tracking analysis. Data are mean ± s.e.m., analyzed using a two-tailed, unpaired Student’s t-test, with n = 4 independent experiments for HCT116 and n = 3 independent experiments for MDA-MB-231. (b) EVs protein amount (μg) was measured by BCA analysis and normalized to cell number at the time of collection. Data are mean ± s.e.m., analyzed using a two-tailed, unpaired Student’s t-test, with n = 6 independent experiments for HCT116 and n = 5 independent experiments for MDA-MB-231. (c) Venn diagram showing unique and overlapping proteins identified by MS/MS between control and APAF1 KO EVs in each cell line. (d-g) EVs were isolated from control and NCF1 KO cells. (d) Transmission electron microscopy for EVs, representative of n = 3 independent experiments. Scale bar: 200 nm. (e) Mode diameter of EVs by nanoparticle tracking analysis. Data are mean ± s.e.m., analyzed using a two-tailed, unpaired Student’s t-test, with n = 4 independent experiments for HCT116 and n = 3 independent experiments for MDA-MB-231 from independent experiments. (f) EVs protein amount (μg) was measured by BCA analysis and normalized to cell number at the time of collection. Data are mean ± s.e.m., analyzed using a two-tailed, unpaired Student’s t-test, with n = 6 independent experiments for HCT116 and n = 5 independent experiments for MDA-MB-231 from independent experiments. (g) EV-DNA was extracted and quantified using Qubit3, normalized to EV protein content. Data are mean ± s.e.m., analyzed using a two-tailed, unpaired Student’s t-test, with n = 5 independent experiments per group. (h) Genomic DNA and EV-DNA were isolated from HCT116 or MDA-MB-231 cells expressing control or NCF1 KO sgRNAs. qPCR was used to detect sgRNA levels in gDNA and EV-DNA and normalized to control sgRNA sequence in gDNA. Data are mean ± s.e.m., analyzed using a two-tailed, unpaired Student’s t-test, n = 5 independent experiments per group from independent experiments. (i) Validation of APAF1 knockdown in NCF1 KO cell lines, the data represent two independent experiments. (j) EV-DNA was extracted from EVs derived from the indicated cell lines. Data are mean ± s.e.m., analyzed using a two-tailed, unpaired Student’s t-test, with n = 4 independent experiments per group.
