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. 2018 May 24;115(24):6279–6284. doi: 10.1073/pnas.1802184115

Fig. 4.

Fig. 4.

d-limonene reduces apoptosis. (A, Left) Heatmap of the apoptotic RNA signature of WT and Aldh3a1−/− EpCAM+ cells from murine SMGs 2 wk after 30-Gy IR (6 Gy/d). (Right) Heatmap of the apoptotic RNA signature of WT SMG EpCAM+ cells 2 wk after 30-Gy IR (6 Gy/d) isolated from mice treated or not treated with daily 10% d-limonene starting 7 d before IR. (B) Percentage of early (annexin V+PI−) and late (annexin V+PI+) apoptotic nonirradiated EpCAM+ WT and Aldh3a1−/− SMG cells by FACS analysis (n = 3; bars indicate SEM; *P < 0.05). (C, Left) Percentage of early and late apoptotic WT EpCAM+ SMG cells treated with 100 μM d-limonene or vehicle (PEG-400) control 18 h after 4-Gy IR by FACS analysis (n = 5–6; bars indicate SEM; **P < 0.01; ***P < 0.001). (Center) FACS dot-plot for control vehicle-treated cells. (Right) FACS dot-plot for d-limonene–treated cells. (D, Left) Representative images of cleaved caspase-3 (red) and DAPI (blue) staining of fixed SMGs removed immediately after the final fraction of 30-Gy IR (6 Gy/d). The treatment group began 10% d-limonene 7 d before IR. (Scale bars: 50 μm.) (Right) The number of cleaved caspase-3+ cells (red) per field blindly counted (n = 6–7 SMGs, 18 images; bars indicate SEM; ***P < 0.001).