Extended Data Fig. 4: Generation of APAF1 KO in murine cancer cells.
(a-f) APAF1 was knocked out in CT26 cells, and EVs were isolated from control and APAF1 KO CT26 cells. (a) Total cell extracts were prepared from WT and APAF1 KO cells, and the lysates were analyzed by Western blot using the indicated antibodies. (b) EVs were analyzed by transition electron microscopy (TEM). Scale bar: 200 nm. (c) Nanoparticle tracking analysis; the histogram represents 3 independent readings per EV sample and cell type. (d) EV protein content was measured using BCA analysis and normalized to the number of cells at the time of collection. Data are mean ± s.e.m., analyzed using a two-tailed, unpaired Student’s t-test, with n = 3 independent experiments per group. (e) EV-DNA was extracted and quantified using Qubit3, normalized to EV protein content. Data are mean ± s.e.m., analyzed by two-tailed, unpaired Student’s t-test, with n = 3 independent experiments per group. (f) EV-DNA was analyzed using chip-based capillary electrophoresis (Bioanalyzer). (g-l) APAF1 was knocked out in KPC cells, and EVs were isolated from control and APAF1 KO KPC cells. (g) Total cell extracts were prepared from WT and APAF1 KO cells, and the lysates were analyzed by Western blot using the indicated antibodies. (h) EVs were visualized by transition electron microscopy. Scale bar: 200 nm. (i) Nanoparticle tracking analysis; the histogram represents 3 independent readings per EV sample and cell type. (j) EV protein content (mg) was measured by BCA analysis and normalized to the number of cells at the time of collection. Data are mean ± s.e.m., analyzed by a two-tailed, unpaired Student’s t-test, with n = 10 per cell type from 5 independent experiments. (k) EV-DNA was extracted and quantified using Qubit3, normalized to protein content. Data are mean ± s.e.m., analyzed by two-tailed, unpaired Student’s t-test, with n = 7 per cell type from 4 independent experiments. (l) EV-DNA was analyzed using chip-based capillary electrophoresis (Bioanalyzer).
