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. 2016 Apr 11;6:23775. doi: 10.1038/srep23775

Figure 14. Disturbed expression of the constitutive Nrf1 mRNA and proteins in the human HCC and relevant cell lines.

Figure 14

(a) Total RNAs were isolated from five distinct HCC cell lines together with the non-cancerous HL7702 cells (all without MG132) and then subjected to RT-qPCR of Nrf1, as described in Fig. 12. The results were calculated as a fold change (mean ± S.E) of Nrf1 in these samples, relative to their respective internal control levels. Significant decreases (*p < 0.05, **p < 0.01, n = 9) of Nrf1 mRNA expression in HCC cells are indicated as compared to the level measured from HL7702 cells. (b) Total lysates (30 μg of proteins) of the cells that had been treated with or without MG132, were subjected to electropherotic separation by 8% SDS-PAGE, followed by western blotting with anti-Nrf1 antibodies. The intensity of immunoblots of Nrf1α (and its derivates) and Nrf1β was quantified as described in Fig. 13, with a ratio of ~140-kDa, ~120-kDa, ~100-kDa and ~65-kDa proteins being calculated by normalization to the 140-kDa value measured from HL7702 cells. (c) Total RNAs were isolated from all seven pairs of the human carcinoma and para-carcinoma tissues that had been confirmed by histopathological examinations (see Fig. S6). The results of RT-qPCR were calculated as a fold change (mean ± S.E) of Nrf1 in these paired samples, relative to their respective internal control levels. Significant decreases (*p < 0.05, **p < 0.01, n = 9) or significant increases ($p < 0.05, $$p < 0.01, n = 9) of Nrf1 mRNA expression in the HCC samples are determined by comparison to its levels measured from corresponding para-carcinoma tissues. (d) Equal amounts (50 μg) of protein extracts from the above carcinoma and para-carcinoma tissues were analyzed by western blotting with anti-Nrf1 antibodies. The intensity of immunoblots was quantified and the results are shown on the bottom with a ratio of ~140-kDa, ~120-kDa and ~65-kDa proteins in each sample (cf. para-carcinoma with carcinoma). (e) Two human HCC samples were visualized by immunohistochemistry with purified anti-Nrf1 antibodies. The areas of carcinoma nodules, invasive borders and pericarcinoma were roughly illustrated in the images that are a representative of at least three independent experiments undertaken on separate occasions.