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. 2019 Oct 22;10:1238. doi: 10.3389/fphar.2019.01238

Figure 3.

Figure 3

Pterostilbene induces cytoplasmic vacuolation in cholangiocarcinoma (CCA) cell lines in a caspase- and apoptosis-independent manner. (A) HCCC-9810 and RBE cells were treated with 30, 60 and 120 µM pterostilbene for 48 h. Microscopic observation revealed the formation of vacuoles in the cytoplasm of treated cells. (B) Transmission electron micrograph (10,000×) of HCCC-9810 and RBE cells treated with 30, 60, and 120 µM pterostilbene for 48 h. Inset shows cell with larger vacuoles after 48 h treatment with pterostilbene. (C, D) HCCC-9810 and RBE cells were treated with different concentrations of pterostilbene for 48 h, stained with Annexin V-FITC and PI, and then measured by flow cytometry. The percentages of cells in early- and late-state apoptosis, as well as total apoptotic cells, after treatment with 0, 30, 60 and 120 µM of pterostilbene, compared to those in control cells, are shown as mean ± SD of three independent experiments. (E) Histograms show the viability of CCA cells treated with DMSO, pterostilbene (30, 60, 120 µM), and/or Z-AVD-FMK for 48 h. Cell viability was determined by a CCK assay after 48 h. (F) Images (200×) show that Z-VAD could not change pterostilbene-induced cytoplasmic vacuolation in cells. Data represent the mean ± SD (**P < 0.01, ***P < 0.001, ****P < 0.0001, n = 3).