Fig. 2.

CANA treatment reduced the proliferation of MSCs and downregulated CCND1 expression. (a) MSCs were incubated with CANA (5 or 10 μM) for the indicated durations, and then the medium was replaced with fresh CANA-free medium. The 0.1% DMSO-treated group served as the control (CTRL). The proliferation of MSCs was evaluated by a cell counting kit-8 (CCK-8) assay (n = 3). (b) MSCs were incubated with CANA (10 μM) and DAPA (10 μM) for the indicated durations, and the medium was then replaced with fresh CANA- and DAPA-free medium. The proliferation of MSCs in each group was detected as described in (a) (n = 3). (c) MSCs treated with CANA (5 or 10 μM, 48 h) were labelled with PI followed by cell cycle analysis by FACS. The percentage of S phase cells was calculated for each group, and the data are shown in a bar graph (n = 3). (d) MSCs were treated with CANA as described in (c) and were incubated with EdU followed by fluorescence detection by microscopy. The percentage of cells stained by EdU was calculated for each group, and the data are shown in a bar graph (n = 3), scale bar: 100 μm. (e) MSCs were treated with 10 μM CANA for 0, 5, 15, 30, 60, 180, and 360 min, and the protein expression levels of ACC, phosphorylated ACC (p-ACC), AMPKα, and phosphorylated AMPKα (p-AMPKα) were evaluated using SDS-PAGE. β-actin served as an internal reference. Quantification of protein expression is presented as the ratio of p-ACC/ACC and p-AMPKα/AMPKα, and the data are shown in a bar graph (n = 3). (f) MSCs were treated as indicated in (e), and the protein expression levels of CCNA2 and CCND1 were analysed using SDS-PAGE. Quantification of protein expression is presented as the ratio of CCNA2/β-actin and CCND1/β-actin, and the data are shown in a bar graph (n = 3). Data are means ± SDs, *P < 0.05; **P < 0.01, ***P < 0.001, ****P < 0.0001.