Fig 1: EPO is inversely correlated to hCAR expression in HCC and promotes proliferation of HepG2 and Hep3B cells.A, hCAR and EPO gene expression was inversely correlated in patients with HCC. A total of 381 HCC samples were analyzed from a publicly accessible TCGA database through SurvExpress, and the overall survival was compared between low-hCAR–expressing (123), medium-hCAR–expressing (232), and high-hCAR expressing (26) groups. B, correlation analysis between hCAR and EPO reveal a Spearman’s correlation coefficient of −0.20 (p < 0.01). C, the pair of low hCAR with high EPO is associated with poor survival. D, in HepG2-hCAR and Hep3B-hCAR cells, treatment with Dox resulted in increased expression of hCAR and decreased expression of EPO in a concentration-dependent manner. E, expression of EPO in HepG2 cells was efficiently abolished after EPO knockdown via lentivirus shRNA targeting different regions of EPO (shEPO 1 to shEPO 5). F, depletion of EPO in HepG2 and Hep3B cells results in suppression of cell growth in a time-dependent manner. HepG2 and Hep3B cells were also visualized by Coomassie blue staining 8 days after infection with lentivirus shEPO 1, shEPO 3, or shCon. G, relative cell growth rate of HepG2-hCAR cells treated with Dox was partially rescued by addition of the recombinant human EPO treatment. Results are expressed as mean ± SD from three independent experiments in D–G. ##p < 0.01; ∗p < 0.05; and ∗∗p < 0.01. Dox, doxycycline; EPO, erythropoietin; hCAR, human constitutive androstane receptor; HCC, hepatocellular carcinoma; TCGA, The Cancer Genome Atlas.
Fig 2: Overexpression of hCAR represses EPO downstream signaling in HepG2 and Hep3B cells.A, schematic illustration of the EPO–EPOR signaling pathway. B and C, HepG2-hCAR and Hep3B-hCAR cells were treated with vehicle control or Dox (1 μg/ml) for 72 h before Western blotting analysis of total and phosphorylation of STAT3 (p-STAT3-Y705), AKT (p-AKT-S473), and ERK1/2 (p-ERK1/2-T183/Y185). D, relative blot densitometry was quantified using ImageJ from three separately prepared cell experiments and normalized to the density of the loading control. Data are expressed as mean ± SD (n = 3). ∗p < 0.05 and ∗∗p < 0.01. Dox, doxycycline; EPO, erythropoietin; EPOR, EPO receptor; ERK1/2, extracellular signal–regulated kinase 1/2; hCAR, human constitutive androstane receptor; STAT3, signal transducer and activator of transcription 3.
Fig 3: Detection of urinary Epo and ESAs by Western blotting with deglycosylation. Urinary Epo and ESAs were investigated by Western blotting. Endogenous urinary Epo (②) was smaller than ESAs (③-⑥). The bands for endogenous Epo and ESAs, except epoetin β pegol, were shifted to 22 kDa by deglycosylation. The band of PEG-bound epoetin β pegol was slightly reduced in size by deglycosylation. The sizes of endogenous Epo and ESAs before and after deglycosylaton by Western blotting and by LC/MS analysis (Table 1) were summarized in the Table. Glycosylated rhEpo (①), urine Epo (②) and ESAs (③-⑥) are indicated by blue arrows. The amount of samples are as follows: urine Epo (33.3 mIU); epoetin α and β (16.7 mIU); darbepoietin (111 pg); and epoetin β pegol 231 pg rhEpo, recombinant human Epo, Endo Epo, endogenous Epo. ① rhEpo, ② human urine Epo, ③ epoetin α, ④ epoetin β, ⑤ darbepoetin α, and ⑥ epoetin β pegol.
Fig 4: Effects of fludrocortisone and ATII on Epo protein expression in the kidney and liver. (a) Effects of fludrocortisone on Epo production in the kidney. Fludrocortisone increased deglycosylated Epo protein expression at 2 and 4 h after the injection. The levels returned to basal values after 6 h. (b) ATII dose-dependently increased deglycosylated Epo protein expression in the renal cortex and outer medulla. The effects were larger in the renal cortex than in the renal outer medulla. (c) ATII increased deglycosylated Epo production in the renal cortex by 1.6- and 3.4-fold by 1 and 10 mg/kg, respectively. (d) In contrast, deglycosylated Epo production was not observed in control and ATII-injected livers. Recombinant rat Epo is shown as the control in Figure 3c. Glycosylated Epo and deglycosylated rat Epo were used in lane 1. Glycosylated Epo and deglycosylated Epo were used in lanes 11 and 12, respectively. * p < 0.05, ** p < 0.01 by a non-parametric analysis using the Kruskal–Wallis test and multiple comparisons by the Shirley–Williams’ test.
Fig 5: Detection of urine Epo and ESAs by Western blotting. (left panel) Glycosylated and deglycosylated epoetin α (38–42 and 22 kDa, respectively) were investigated by Western blotting. The detection limit was improved by deglycosylation. (right panel) Urinary Epo and epoetin α without deglycosylation were investigated by Western blotting. Urinary Epo was smaller than epoetin α. rhEpo; recombinant human Epo, deg-rhEpo; deglycosylated recombinant human Epo.
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