Fig 1: The functional model of Snail-Nur77-PEPCK1 pathway.In HCC, PEPCK1 was sumoylated and p300 enhanced the sumoylation through acetylating Ubc9. Sumoylation led to PEPCK1 degradation via ubiquitination way, consequently boosted glycolysis and suppressed gluconeogenesis, promoted HCC development. Nur77 could stabilize PEPCK1 by competing Ubc9 binding to PEPCK1. Nevertheless, Snail with other factors suppressed expression of Nur77 gene through DNA methylation and histone deacetylation on Nur77 promoter.
Fig 2: Activation of Nur77 attenuates PEPCK1 sumoylation and stabilizes PEPCK1.(a) Nur77 abolished SUMO1/Ubc9-induced sumoylation (top), and p300-enhanced sumoylation (bottom) in different liver cancer cell lines. The amount of PEPCK1 sumoylation was quantitated by software Image J and presented under lanes of PEPCK1. (b) The role of Nur77 in elevating endogenous PEPCK1 expression levels in different liver cancer cell lines that were transfected with SUMO1 and Ubc9. (c) Nur77 elevated PEPCK1 protein expression in a time-dependent manner in HepG2 cells that were transfected with SUMO1 and Ubc9. (d) Nur77 blocked Ubc9 binding to PEPCK1 in 293T cells detected by co-IP assay. (e) Nur77 impaired p300 acetylation effect on PEPCK1 (left) or Ubc9 (right) detected with specific anti-acetylated lysine antibody (Ac) in 293T cells. (f) Images (left) and weight (right) of xenograft tumour in nude mice (n=6). PEPCK1 or PEPCK1 K124R was stably overexpressed in Huh7 cells, and then Nur77 was further stably transfected into these cells. Cells were injected subcutaneously into the posterior flanks of nude mice. Scale bars, 1 cm. NEM (20 mM) was added to cell lysates for repression of de-sumoylation in each sumoylation assay. GAPDH was used to indicate the amount of loading proteins. Data were represented as means±s.e.m. of mice in the number indicated in the parenthesis. *P<0.05; ***P<0.001. The data were analysed using one-way ANOVA followed by Tukey post hoc test.
Fig 3: HucMDEs improve the glucose and lipid metabolism by promoting autophagy. a L-O2 cells were treated with PA, PA + HucMDEs, PA + 3-MA, or PA+ HucMDEs + 3-MA. Protein levels of MAP 1LC3B were examined by Western blotting. Glycolysis-related proteins (GCK, PFK, PK) (b), glycogen synthesis-related proteins (p-GSK3ß, GSK3ß) (c), and gluconeogenesis-related proteins (G-6-P, PEPCK) (d) in L-O2 cells of the indicated groups were detected by Western blotting. e PAS staining of L-O2 cells in the indicated groups. f SREBP-1c and PPARa in L-O2 cells of the indicated groups were detected by Western blotting. All results are expressed as the mean ± SD (*P < 0:05; **P < 0:01; ***P < 0.001)
Fig 4: Sumoylation induces PEPCK1 degradation.(a) In DEN/CCl4-induced HCC samples (left) or normal livers (Ctrl) versus DEN/CCl4-induced HCC samples (right), endogenous PEPCK1 was first immunoprecipitated and then incubated with anti-PEPCK1 antibody or anti-SUMO1 antobody, respectively. Asterisk represents position of PEPCK1 protein; arrowheads indicate sumoylation bands. (b) The levels of PEPCK1 and its sumoylation in clinical carcinoma and paired para-carcinoma samples. Endogenous SUMO1 was immunoprecipitated and then incubated with anti-PEPCK1 antibody. (c) Top, HepG2 cell lysates with or without NEM (20 mM) was immunoprecipitated with anti-SUMO1 antibody and then incubated with anti-PEPCK1 antibody. Bottom, SUMO1 and Ubc9 were transfected into 293T cells, and Flag-SUMO1 was immunoprecipitated and then detected by western blot with anti-PEPCK1 antibody. (d) Sumoylation effect on endogenous PEPCK1 expression in different liver cancer cell lines that were transfected with SUMO1/Ubc9 (top) or treated with sumoylation inhibitor, anacardic acid (20 µM, 12 h) with or without SUMO1/Ubc9 transfection (middle and bottom). (e) Sumoylation attenuated PEPCK1 stability in SMMC-7721 cells that were transfected with different plasmids and then treated with CHX (100 µg ml-1) for different times (left). The amount of PEPCK1 protein was quantitated by software Image J (right). (f) Determination of the critical sites for PEPCK1 sumoylation in SMMC-7721 cells. The amount of PEPCK1 protein was normalized in each panel. (g) Lys124 site was important to stabilize PEPCK1 protein in SMMC-7721 cells with CHX (100 µg ml-1) treatment. (h) Sumoylation-induced PEPCK1 degradation via ubiquitination pathway in SMMC-7721 cells that were treated with MG132 (5 µM) or ALLM (20 µM) for 12 h. The expression level of PEPCK1 (left) or ubiquitination levels of PEPCK1, PEPCK1K124R and PEPCK1K471&473R (middle and right) were detected. NEM (20 mM) was added to cell lysates for repression of de-sumoylation in each sumoylation assay. Tubulin or GAPDH was used to indicate the amount of loading proteins. Data were represented as means±s.e.m. of at least three independent experiments. *P<0.05; **P<0.01. The data were analysed using one-way ANOVA followed by Tukey post hoc test.
Fig 5: HucMDEs improve glucose and lipid metabolism both in vivo and in vitro. Glycolysis-related proteins (GCK, PFK, and PK) (a), glycogen synthesis-related proteins (p-GSK3ß, GSK3ß) (b), and gluconeogenesis-related proteins (G-6-P, PEPCK) (c) in the livers of the indicated groups were detected by Western blotting. d PAS staining in the livers of the indicated groups. e The hepatic lipid synthesis protein, SREBP-1c, and lipolytic protein, PPARa, in the livers of the indicated groups were detected by Western blotting. Glycolysis-related proteins (GCK, PFK, PK) (f), glycogen synthesis-related proteins (p-GSK3ß, GSK3ß) (g), and gluconeogenesis-related proteins (G-6-P, PEPCK) (h) in L-O2 cell groups of Control + PBS, 0.25 mM PA + PBS, PA + HDEs (30 µg/ml), and PA + HucMDEs (30 µg/ml) were detected by Western blotting. i PAS staining in L-O2 cells. j The hepatic lipid synthesis protein, SREBP-1c, and lipolytic protein, PPARa, in L-O2 cells of the indicated groups were detected by Western blotting. All the results are expressed as the mean ± SD (*P < 0:05; **P < 0:01; ***P < 0.001)
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