Fig 1: KHK-A–mediated p62 S28 phosphorylation promotes hepatocellular tumorigenesis and is associated with clinical aggressiveness of human HCC.(A) Huh7 (1 × 106) cells with or without knock-in of KHK-A S80A (top) or p62 S28A (bottom) expression were intrahepatically injected into athymic nude mice (n = 7 per group). The mice were euthanized and examined for tumor growth 28 days after injection. The arrows point to the tumors. Tumor volumes were calculated (right). Data represent the means ± SD of seven mice. **P < 0.01 by a two-tailed Student’s t test. (B) IHC analyses of tumor tissues were performed with an anti-Ki67 antibody. Ki67-positive cells were quantified in 10 microscope fields. **P < 0.01 by a two-tailed Student’s t test. (C and D) IHC analyses of xenograft tumors from nude mice were performed with the indicated antibodies. (E) IHC staining of 30 human HCC and matched nontumor tissue samples was performed with the indicated antibodies. Representative photos of stains of two cases are shown. (F) IHC staining of 90 human HCC specimens was performed with the indicated antibodies. Representative images from the staining of four different specimens are shown. (G) Kaplan-Meier plots of the overall survival rates in human HCC specimens (n = 90) in groups with high (staining score, 4 to 8) and low (staining score, 0 to 3) expression of KHK-A pS80, p62 pS28, and KHK-C. The P values were calculated using the log-rank test. (H) The mechanism underlying differentiated responses of normal hepatocytes and HCC cells to oxidative stress. Oxidative stress results in AMPK-mediated phosphorylation of KHK-A S80, primarily expressed in HCC cells, but not of KHK-C, primarily expressed in normal hepatocytes. S80-phosphorylated KHK-A acts as a protein kinase and phosphorylates p62 S28, resulting in p62 oligomerization, p62 and Keap1 aggregation, and nuclear translocation and activation of Nrf2, which lead to counteracting ROS production, maintaining tumor cell survival, and promoting HCC development. In contrast, normal hepatocytes, lacking KHK-A–mediated antioxidative response, have high levels of ROS production and increased apoptosis. Ub, ubiquitin.
Fig 2: Western blot analysis in four groups at 2, 4 and 6 week. Representative Western blot bands and relative expression levels for LC3-II, Beclin1, P62, Keap1 and Nrf2 at 2, 4 and 6 weeks after fracture. *p < 0.05 compared to S; #p < 0.05 compared to OVX; &p < 0.05 compared to OH.
Fig 3: Protein expression levels of proinflammation proteins in heart tissue of mice using western blot method. (A) Western blotting analysis and quantitative analysis of (B) Keap1, (C) Nrf2, (D) HO-1, (E) IKK and (F) NF-?B. Data are presented as the mean ± SD. Each experiment was repeated three times independently. *P<0.05 vs. NC group. #P<0.05 vs. OT group. DHE, dehydrocostus lactone; TXNIP, thioredoxin-interacting protein; NC, negative control; OT, DHE treatment group; OI, DHE treatment combined with TXNIP inhibition group; OH, DHE treatment combined with TXNIP overexpression group; TLR, toll-like receptor; HMGB1, high mobility group protein B1; NLRP3, NOD-, LRR- and pyrin domain-containing protein 3; MW, molecular weight.
Fig 4: Immunohistochemistry results for autophagy and Keap1-Nrf2 signaling in the four groups at 6 weeks. At 6 weeks, the autophagy level in the sham group was decreased to a stable state. In the OVX group, high levels of oxidative stress and autophagy were observed, part of the bone tissue was undergoing bone remodeling, and dead bone was also observed at the fracture site. A significant increase in the level of P62 was observed in the OH and OHA groups compared with the OVX group. Furthermore, the expression of Nrf2 in the OH and OHA groups tended to be normal but still high compared with that in the sham group, but compared with that in the OVX group, the level of autophagy was low, and the antioxidative stress response was strong. Scale bar = 1000 µm and 50 µm.
Fig 5: Protein expression levels of HO-1 regulator proteins in H9c2 cells using western blot method. (A) Western blotting analysis and quantitative analysis of (B) Keap1, (C) Nrf2, (D) HO-1, (E) IKK and (F) NF-?B in H9c2 cells. Data are presented as the mean ± SD. Each experiment was repeated three times independently. *P<0.05 vs. NC group. #P<0.05 vs. OT group. DHE, dehydrocostus lactone; TXNIP, thioredoxin-interacting protein; NC, negative control; OT, DHE treatment group; OI, DHE treatment combined with TXNIP inhibition group; OH, DHE treatment combined with TXNIP overexpression group; Nrf2, nuclear factor erythroid 2-related factor 2; HO-1, heme oxygenase-1; Keap1, Kelch-like ECH-associated protein 1; MW, molecular weight.
Supplier Page from Abcam for Anti-Keap1 antibody