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. 2022 Jun 3;10(6):1316. doi: 10.3390/biomedicines10061316

Figure 6.

Figure 6

Rottlerin stimulates exosome/microvesicle release via activation of the AMPK (AMP-activated protein kinase) pathway. (A) Western blotting and densitometric analyses of whole-cell lysates using anti-phospho-AMPK (T172), AMPK (total), and β-actin antibodies in C6 cells exposed to LDL (20 μg/mL of cholesterol) and 2.5 μM U18666A for a total of 16 h without (Control) or with rottlerin (5 μM and 10 μM, Rot) for the last 4 h. Results are the mean ± SEM of three independent experiments. Representative immunoblots are shown. (B) C6 cells were exposed to DiI-LDL (20 μg/mL of cholesterol) and U18666A for a total of 16 h without (Control) or with rottlerin in combination with or without compound C (10 μM or 20 μM, CC) for the last 4 h. The cells were then washed, fixed, and analyzed by confocal microscopy. Images show DiI-LDL in red and nuclei labeled with Hoechst-33342 in blue. Representative results from three independent experiments are shown. Scale bars = 25 μm. (C) The graph shows quantitative results of DiI-LDL labelling (mean intensity, a.u.) in cells of each photomicrograph. Results are the mean ± SEM of three independent experiments, with 8−10 photographs per experiment. (D) Total particle secretion analyzed by nanoparticle tracking. The experiments started with the same number of cells. Cells were exposed to LDL (60 µg/mL of cholesterol) in the presence of 2.5 µM U18666A for 12 h. Then, the medium was removed, cells were washed twice, and serum-free medium was added, supplemented with U18666A, and incubation was continued for 4 h with or without 5 μM rottlerin (Rot) in combination with or without 20 μM compound C (CC). At the end of the incubation, the medium was removed, and the exosomes/microvesicles were isolated and analyzed. The graphs represent the total number of particles per experiment analyzed by nanoparticle tracking. Means ± SEM of three measures. Statistical comparisons shown are rottlerin versus control (* p < 0.05, *** p < 0.001) or compound C plus rottlerin versus rottlerin alone (++ p < 0.01 and +++ p < 0.001).