Figure 2.
EV production increased during neuronal differentiation.(A) Schematic of the EV purification strategy. (B) Representative electron micrographs of negatively stained samples of purified EVs at 9,300× magnification. Purified EVs from untreated PC12 cells cultured for 3 d (PC12-EV) or treated with NGF for 3, 6, and 9 d (N3-EV, N6-EV, and N9-EV). During PC12 differentiation, EVs were collected from 3-d-cultured cells, and fresh medium together with NGF were replaced every 3 d. Scale bar, 0.2 µm. (C) Nanoparticle tracking analysis of the size distribution and the number of purified EVs from 420-ml medium of untreated PC12 cells cultured for 3 d or treated with NGF for 3, 6, and 9 d. (D) The number of EVs released per PC12 cell untreated or treated with NGF for 3, 6, and 9 d. EV number was quantified by nanoparticle tracking analysis. Cell number was quantified with a hemocytometer. The values represent the mean ± SD, from three independent experiments. Error bars represent SD from independent samples. (E) The number of EVs released per N2A cell cultured for 3 d or treated with RA for 3 and 6 d. During N2A differentiation, EVs were collected from 3-d-cultured cells, and fresh medium together with RA were replaced every 3 d. The values represent the mean ± SD, from three independent experiments. Error bars represent SD from independent samples. (F) The number of EVs released per ESC untreated or differentiated for 8 and 12 d. During ES differentiation, EVs were collected from 2-d-cultured cells, and fresh medium was replaced every 2 d. The values represent the mean ± SD, from two independent experiments. Error bars represent SD from independent samples. (G) Immunoblots of CD9, Hsc70, Flot2, CD63, Alix, and Tsg101 in EVs from the same number of cells. 2 × 107 PC12 cells were untreated or treated with NGF for 3, 6, and 9 d. (H) Quantitative analysis of the immunoblots in G. The values represent the mean ± SD, from two independent experiments. Error bars represent SD from independent samples. The signal in PC12-EV group was set as 1. (I) Immunoblots of CD9, Hsc70, Flot2, CD63, Alix, and Tsg101 in EVs from the same number of cells. 2 × 107 N2A cells were untreated or treated with RA for 3 and 6 d. (J) Quantitative analysis of the immunoblots in I. The values represent the mean ± SD, from two independent experiments. Error bars represent SD from independent samples. The signal in N2A-EV group was set as 1.

EV production increased during neuronal differentiation. (A) Schematic of the EV purification strategy. (B) Representative electron micrographs of negatively stained samples of purified EVs at 9,300× magnification. Purified EVs from untreated PC12 cells cultured for 3 d (PC12-EV) or treated with NGF for 3, 6, and 9 d (N3-EV, N6-EV, and N9-EV). During PC12 differentiation, EVs were collected from 3-d-cultured cells, and fresh medium together with NGF were replaced every 3 d. Scale bar, 0.2 µm. (C) Nanoparticle tracking analysis of the size distribution and the number of purified EVs from 420-ml medium of untreated PC12 cells cultured for 3 d or treated with NGF for 3, 6, and 9 d. (D) The number of EVs released per PC12 cell untreated or treated with NGF for 3, 6, and 9 d. EV number was quantified by nanoparticle tracking analysis. Cell number was quantified with a hemocytometer. The values represent the mean ± SD, from three independent experiments. Error bars represent SD from independent samples. (E) The number of EVs released per N2A cell cultured for 3 d or treated with RA for 3 and 6 d. During N2A differentiation, EVs were collected from 3-d-cultured cells, and fresh medium together with RA were replaced every 3 d. The values represent the mean ± SD, from three independent experiments. Error bars represent SD from independent samples. (F) The number of EVs released per ESC untreated or differentiated for 8 and 12 d. During ES differentiation, EVs were collected from 2-d-cultured cells, and fresh medium was replaced every 2 d. The values represent the mean ± SD, from two independent experiments. Error bars represent SD from independent samples. (G) Immunoblots of CD9, Hsc70, Flot2, CD63, Alix, and Tsg101 in EVs from the same number of cells. 2 × 107 PC12 cells were untreated or treated with NGF for 3, 6, and 9 d. (H) Quantitative analysis of the immunoblots in G. The values represent the mean ± SD, from two independent experiments. Error bars represent SD from independent samples. The signal in PC12-EV group was set as 1. (I) Immunoblots of CD9, Hsc70, Flot2, CD63, Alix, and Tsg101 in EVs from the same number of cells. 2 × 107 N2A cells were untreated or treated with RA for 3 and 6 d. (J) Quantitative analysis of the immunoblots in I. The values represent the mean ± SD, from two independent experiments. Error bars represent SD from independent samples. The signal in N2A-EV group was set as 1.

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