Fig 1: Western blot analysis of ROX-induced Epo production in the body. (a) Epo protein expression in ROX-treated male rat. 1, Salivary gland; 2, thymus; 3, lung; 4, heart; 5, liver; 6, pancreas; 7, spleen; 8, adrenal gland; 9, kidney (cortex); 10, testes; and 11, epididymis. (b) Epo protein expressions in salivary gland, thymus, liver, spleen and kidney were compared in control and ROX-treated male rats. (c) Epo protein abundances in lung, pancreas, kidney, testis and epididymis were compared in control and ROX-treatedmale rats. (d) Epo protein expression in ROX-treated female rat. 1, Salivary gland; 2, thymus; 3, lung; 4, heart; 5, liver; 6, pancreas; 7, spleen; 8, adrenal gland; 9, kidney (cortex); and 10, ovary. (e) Epo protein expressions in salivary gland, thymus, liver, spleen, kidney and ovaries were compared in control and ROX-treated female rats. (f) Summarized data. Epo protein expression was normalized by measuring the β-actin expression. * indicates p < 0.05, n = 3–6. 1, Olfactory bulb; 2, cerebrum; 3, salivary gland; 4, thymus; 5, lung; 6, heart; 7, liver; 8, pancreas; 9, spleen; 10, adrenal gland; 11, kidney (cortex); 12, epididymis; and 13, ovary. a, Control and b, ROX.
Fig 2: Effects of ROX on Epo mRNA and protein expression in the kidneys and liver. Effects of ROX on Epo (a), HIF2α (b), HIF1α (c) and PHD2 (d) mRNA expressions. (e) Effects of ROX on Epo protein expressions in the kidney (left half) and the liver (right half). * indicates p < 0.05. n = 3–9. CK, R5K and R10K show the control, R5- and R10-treated rat kidney, respectively. CL, R5L and R10L show the control, R5- and R10-treated rat liver, respectively.
Fig 3: Plasma Epo concentration and Western blot analysis of serum Epo. (a): Plasma Epo concentration in control and ROX (50 mg/kg)-injected rats. ROX significantly increased the plasma Epo concentration from 1.2 ± 0.1 to 1072 ± 333 mIU/mL (n = 5–6, p < 0.001). (b): Serum Epo was examined by Western blot analysis as described in the methods. Plasma Epo concentration in control, R5 (50 mg/kg) and R10 (100 mg/kg) rats was 1.4, 2696 and 3200 mIU/mL, respectively. Glycosylated Epo (Epo) and deglycosylated Epo (degEpo) were detected at 35–38 and 22 kDa, respectively.
Fig 4: Comparison of glycosylated and deglycosylated Epo proteins by Western blotting. Lanes 1–5 show the same amount of glycosylated (PNGase (−)) and deglycosylated (PNGase (+)) Epo. Epo; (glycosylated) erythropoietin, degEpo; deglycosylated Epo, rRatEpo; recombinant rat Epo. Glycosylated and deglycosylated recombinant rat Epo were combined.
Fig 5: Immunohistochemical analysis of the effects of ROX on Epo production by the kidneys. Under control conditions, Epo staining was found from the proximal tubule to the collecting duct (a). ROX increased Epo production in the peritubular cells around proximal tubules after 6 h (R10 > R5). Red arrowheads show Epo positive peritubular cells ((d–f) and red square of (a–c)). Epo staining was not increased in the distal tubules but slightly increased in the proximal tubules by ROX ((g–i) and yellow square of (a–c)). Scale bar: 20 µm (a–c) and 10 µm (d–i). PCT, proximal convoluted tubules; and CNT, connecting tubules.
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