Fig. 1. APG increased the sensitivity of BEL-7402/ADM cells to ADM

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Fig. 1. APG increased the sensitivity of BEL-7402/ADM cells to ADM Fig. 1. APG increased the sensitivity of BEL-7402/ADM cells to ADM. ( A – C ) MTT assays. Cells were incubated for 48h with increasing concentrations of APG (2.5–80 μM) (A), ADM (0.5–8 μM) (B) or with 10 μM of APG followed by 48h exposure to ADM (0.5–8 μM) (C). * P < 0.05, ** P < 0.01 compared with ADM alone-treated group. ( D ) Cells were treated with 20 μM of APG or/and 2 μM of ADM for 24h. The treated cells were subjected to cell cycle analysis. * P < 0.05, ** P < 0.01 versus untreated control cells; <sup>##</sup>P < 0.01 versus cells with ADM treatment alone. ( E ) Cells were treated with 20 μM of APG or/and 2 μM of ADM for 24h. Fluorescent staining of nuclei by DAPI. Condensed and fragmented nuclei and apoptotic bodies were indicated with arrows. All images are shown at ×200. From: Apigenin sensitizes doxorubicin-resistant hepatocellular carcinoma BEL-7402/ADM cells to doxorubicin via inhibiting PI3K/Akt/Nrf2 pathway Carcinogenesis. 2013;34(8):1806-1814. doi:10.1093/carcin/bgt108 Carcinogenesis | © The Author 2013. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Fig. 2. APG increased intracellular accumulation of ADM in BEL-7402/ADM cells. ( A ) BEL-7402/ADM cells were treated with ADM (8 μM) for 3h following 24h of exposure to APG (10 and 20 μM). The fluorescence intensity of intracellular ADM was directly observed by fluorescence microscopy. All images are shown at ×200. ( B ) Intracellular accumulation of ADM analyzed by FCM. Results are presented as fold change in fluorescence intensity relative to control BEL-7402 cells. <sup>##</sup>P < 0.01 versus parental + ADM; * P < 0.05, ** P < 0.01 versus resistance + ADM. ( C ) Effect of APG on the accumulation of Rh123 in BEL-7402/ADM cells. Cells were incubated with 5 μg/ml of Rh123 for 1h following 24h of exposure to APG (10 and 20 μM). Data are expressed as mean ± SD ( n = 3). <sup>##</sup>P < 0.01 versus parental + Rh123; * P < 0.05, ** P < 0.01 versus resistance + Rh123. From: Apigenin sensitizes doxorubicin-resistant hepatocellular carcinoma BEL-7402/ADM cells to doxorubicin via inhibiting PI3K/Akt/Nrf2 pathway Carcinogenesis. 2013;34(8):1806-1814. doi:10.1093/carcin/bgt108 Carcinogenesis | © The Author 2013. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Fig. 3. Dose-dependent effect of APG on mRNA and nuclear protein expression of Nrf2, HO-1, AKR1B10 and MRP5 in BEL-7402/ADM cells. ( A ) Cells were treated with APG (10 and 20 μM) for 24h and subjected to real-time quantitative PCR. ( B ) BEL-7402/ADM cells were treated with APG (10 and 20 μM) for 24h prior to western blot. Data are expressed as mean ± SD ( n = 3). <sup>#</sup>P < 0.05, <sup>##</sup>P < 0.01 versus parental; * P < 0.05, ** P < 0.01 versus resistance. From: Apigenin sensitizes doxorubicin-resistant hepatocellular carcinoma BEL-7402/ADM cells to doxorubicin via inhibiting PI3K/Akt/Nrf2 pathway Carcinogenesis. 2013;34(8):1806-1814. doi:10.1093/carcin/bgt108 Carcinogenesis | © The Author 2013. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Fig. 4. APG increased the sensitivity of BEL-7402/ADM cells to ADM by inhibition of Nrf2. ( A ) Effect of Nrf2 knockdown on drug-resistant gene protein expression. * P < 0.05, ** P < 0.01 versus resistance. ( B ) Effect of Nrf2 knockdown on the sensitivity to ADM. Cells were pretreated with 10 μM of APG for 24h prior to 48h exposure to ADM (0.5–8 μM). Cell viability was monitored by MTT assay. Data are expressed as mean ± SD ( n = 3). From: Apigenin sensitizes doxorubicin-resistant hepatocellular carcinoma BEL-7402/ADM cells to doxorubicin via inhibiting PI3K/Akt/Nrf2 pathway Carcinogenesis. 2013;34(8):1806-1814. doi:10.1093/carcin/bgt108 Carcinogenesis | © The Author 2013. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Fig. 5. Inhibition of PI3K reduced Nrf2-mediated response in BEL-7402/ADM cells. ( A ) APG suppressed p-Akt in BEL-7402/ADM cells. Reduced expression of p-Akt and increased expression of p-P38, p-JNK and p-ERK1/2 were observed after exposure to APG (20 μM) for 24h. <sup>##</sup>P < 0.01 versus parental; * P < 0.05, ** P < 0.01 versus resistance. ( B ) LY294002 (50 μM) reduced the protein levels of nuclear Nrf2 and Nrf2-target genes including HO-1, AKR1B10 and MRP5. * P < 0.05, ** P < 0.01 versus resistance. ( C ) BEL-7402/ADM cells were pretreated with LY294002 (50 μM) or/and APG for 24h prior to fluorescence microscopy. All images are shown at ×200. From: Apigenin sensitizes doxorubicin-resistant hepatocellular carcinoma BEL-7402/ADM cells to doxorubicin via inhibiting PI3K/Akt/Nrf2 pathway Carcinogenesis. 2013;34(8):1806-1814. doi:10.1093/carcin/bgt108 Carcinogenesis | © The Author 2013. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Fig. 6. APG sensitized BEL-7402 xenografts to ADM treatment Fig. 6. APG sensitized BEL-7402 xenografts to ADM treatment. ( A ) Combined treatment with APG and ADM significantly reduced tumor weight. When tumor size reached 100 mm <sup>3</sup> , mice (eight per group) were treated with vehicle, ADM (3mg/kg), APG (50mg/kg) or both APG and ADM through intraperitoneally every 3 days for a total of seven times. Tumors were excised and weighed at the end of the experiment (40 days). ( B ) Combined treatment with APG and ADM significantly inhibited tumor growth. Tumor sizes were measured semiweekly. ( C ) APG reduced Nrf2 expression in xenografts in vivo . * P < 0.05, ** P < 0.01 versus vehicle control; <sup>#</sup>P <0.05, <sup>##</sup>P <0.01 vs combination. From: Apigenin sensitizes doxorubicin-resistant hepatocellular carcinoma BEL-7402/ADM cells to doxorubicin via inhibiting PI3K/Akt/Nrf2 pathway Carcinogenesis. 2013;34(8):1806-1814. doi:10.1093/carcin/bgt108 Carcinogenesis | © The Author 2013. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com