1. Volume 136, Issue 1, Pages 341-350.e8 (January 2009)
Suppressive Effects of Retinoids on Iron-Induced Oxidative Stress in the Liver 
Hiroyuki Tsuchiya, Yuji Akechi, Remina Ikeda, Ren Nishio, Tomohiko Sakabe, Kei Terabayashi, Yoshiaki Matsumi, An Afida Ashla, Yoshiko Hoshikawa, Akihiro Kurimasa, Takao Suzuki, Naoto Ishibashi, Shingo Yanagida, Goshi Shiota 
Gastroenterology 
Volume 136, Issue 1, Pages e8 (January 2009)
DOI: /j.gastro
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2. Figure 1
Iron metabolism in RAR-E Tg mouse. (A) RT-PCR analysis of hemojuvelin (Hjv) expression in the livers of 8-month old RAR-E Tg and wild-type mice. Gapdh is shown as an internal control. M, marker. (B) RT-PCR analysis of expression levels of iron metabolism-related genes in the livers of 8-month-old RAR-E (filled bars) and wild-type (open bars) mice. The relative expression levels to gapdh of Tg mice were normalized by that of wild type mice. hepc, hepcidin; fpn, ferroportin; hjv, hemojuvelin; tf, transferrin; tfr1, transferrin receptor 1; tfr2, transferrin receptor 2; fth, ferritin heavy chain; ftl, ferritin light chain. (C) Non-heme iron content in RAR-E (filled bars) and wild type (open bars) mouse livers. Data are shown as mean ± SD (n = 4–6). *P < .05, **P < .01, ***P < .001 vs wild type mouse.
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3. Figure 2
Effect of retinoids on hemojuvelin (HJV), transferrin receptor 2 (TfR2), hepcidin (HEPC) and ferroportin (FPN) expressions. (A, B, C, and D) Real-time RT-PCR analysis for HJV (A), TfR2 (B), HEPC (C), and FPN (D) expressions. HuH7 cells were treated with 0–20 μM of all-trans retinoic acid (ATRA; filled circles) and NIK-333 (NIK; open circles) for 24 h. Expression levels relative to β-actin were normalized by the control (0 μM retinoid). Data are shown as means ± SD (n = 3). *P < .05, **P < .01, ***P < .001 vs control. (E) Western blot analysis for HJV, TfR2, and FPN, and dot blot analysis for HEPC. Actin is shown as an internal control. Total protein samples were prepared from HuH7 treated with dimethylsulfoxide (control; CTRL) or 5 μM of ATRA and NIK-333 for 24 h.
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4. Figure 3
The factors involved in the regulation of hemojuvelin (HJV) expression. (A) ChIP assay for the 5′-UTR of HJV gene. Nuclear extracts were prepared from HuH7 cells after the treatment with dimethylsulfoxide (C), ATRA (A), or NIK-333 (N) for 6 h. The 10-fold diluted nuclear extracts were used as an input control for PCR (InPut [10%]). The data are representative of 2 separate experiments. (B) Western blot analysis for HJV expression after RARα knockdown. HuH7 cells were transfected with siNC (negative control) or siRARα. Cell lysates were prepared after 48 h. Actin is shown as an internal control. (C and D) Real-time RT-PCR analysis of HJV (C) and RARβ (D) expressions in retinoid signaling-impaired cells. HuH7-derived cell lines expressing RAR-E (black lines) and LacZ (grey lines) were treated with 0–20 μM ATRA for 24 h. Expression levels relative to β-actin were normalized by the control (0 μM ATRA). Data are shown as means ± SD (n = 4). *P < .05, **P < .01 vs LacZ.
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5. Figure 4
Effect of retinoids on iron uptake by HuH7 cells. (A) Transferrin-mediated iron uptake. HuH7 cells were exposed to 50 μM [59Fe]holo-transferrin in the absence (control; CTRL) and presence of 0–20 μM ATRA or NIK-333 for 24 h. Data are shown as means ± SD (n = 3). *P < .05, **P < .01, ***P < .001 vs CTRL. (B) Non-heme iron content in HuH7 cells treated with retinoids and Fe-NTA. The cells were exposed to the indicated concentrations of ATRA (black bars) or NIK-333 (grey bars) in the presence or absence of 20 μM Fe-NTA (Fe) for 96 h. Data are shown as means ± SD (n = 3). *P < .05, **P < .01 vs 20 μM Fe-NTA alone, †P < 0.05, ‡P < .01 vs non-treatment control. (C) Non-heme iron contents in HuH7 (open circles) and HuH-HJV (filled circles). The cells were cultured in the presence of 0, 10, 20, and 50 μM Fe-NTA for 96 h.
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6. Figure 5
Suppression of iron-induced oxidative stress by retinoids. (A) Antioxidant capacity in HuH7 cells. (B) Iron-catalyzed production of hydroxyl radical. (C) Cytotoxic effect of iron to HuH7 cells. HuH7 cells were exposed to the indicated concentrations of Fe-NTA (Fe) in the absence (control; grey filled circles) and presence of 5 μM ATRA (black filled circles) or NIK-333 (open circles) for 96 h. Data are shown as means ± SD (n = 3). *P < .05, **P < .01, ***P < .001 vs control.
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7. Figure 6
Effect of dietary retinoid on iron metabolism and oxidative stress in the liver. (A) A schematic representation of retinoid-deficient and retinoid-excess mice models with iron overload. (B) Non-heme iron concentrations in the livers of mice fed retinoid diets. Inset, non-heme iron concentrations in the livers before iron overload (day 0). (C) The ratio of reduced (GSH) and oxidized glutathione (GSSG) in the livers at day 10 after iron overload. (D) Dot blot analysis for 4-HHE-, MDA-, and 4-HNE-modified protein expressions in the livers at day 10 after iron overload. NC; a negative control experiment without the primary antibodies. (E) Quantification of dot intensities obtained by dot blot analysis shown in (D). −, retinoid-deficient diet; +, normal diet; ++, retinoid-excess diet. Data are shown as means ± SD (n = 5). *P < .05, **P < .01, ***P < .001.
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8. Figure 7
Effect of dietary retinoids on Hjv, Tfr2, Hepc and Fpn expressions in the liver. (A, B, C, and D) Real-time RT-PCR analysis for hemojuvelin (Hjv; A), transferrin receptor 2 (TfR2; B), hepcidin (Hepc; C) and ferroportin (Fpn; D). Data are shown as mean ± SD (n = 5). *P < .05, **P < .01, ***P < Expression levels of genes were normalized by that of β-actin. Square, retinoid-deficient diet; triangles, normal diet; circles, retinoid-excess diet. (E) Western blot analysis for Fpn in the livers at day 10 after iron overload. Actin is shown as an internal control.
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9. Figure 8
Schematic representation of the model describing the retinoid-regulated hepatic iron metabolism. Retinoid transcriptionally inhibits hemojuvelin (HJV) expression, which further induces the down-regulation of transferrin receptor 2 (TfR2) and hepcidin (HEPC), leading to decreased transferrin-mediated iron uptake and increased iron export by ferroportin (FPN). Eventually, the retinoid signaling results in decreased hepatic iron (Fe) content and iron-induced oxidative stress (·OH), possibly by this pathway.
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10. Supplementary Figure 1
Time-course analysis of hemojuvelin (HJV), transferrin receptor 2 (TfR2), and hepcidin (HEPC) expressions by real-time RT-PCR. (A) HJV, (B) TfR2, (C) HEPC expressions. HuH7 cells were in the absence (CTRL; grey filled circles) and presence of 5 μM ATRA (black filled circles) or NIK-333 (open circles) for 24 h. Expression levels of genes were normalized by that of β-actin. Data are shown as means ± SD (n = 3). *P < .05, **P < .01, ***P < .001 vs control.
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11. Supplementary Figure 2
Effect of iron on retinoid-induced downregulation of iron metabolism-related genes expression. (A) HJV, (B) TfR2, (C) HEPC expressions. HuH7 cells were treated with DMSO (CTRL), 5 μM ATRA, or NIK-333 (NIK) in the absence or presence of 20 μM Fe-NTA (Fe) for 24 h, followed by real-time RT-PCR analysis. Expression levels relative to β-actin were normalized by 0 μM Fe CTRL. Data are shown as means ± SD (n = 3). *, **, and *** indicate statistical significance with P < .05, P < .01 and P < .001, respectively, vs CTRL.
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12. Supplementary Figure 3
Effect of retinoids on hemojuvelin (HJV), transferrin receptor 2 (TfR2), and hepcidin (HEPC) expressions in HuH-HJV cells. (A) HJV, (B) TfR2, (C) HEPC expressions. HuH7 and HuH-HJV cells were cultured in the absence (CTRL) and presence of 5 μM ATRA or NIK-333 (NIK) for 24 h, followed by real-time RT-PCR analysis. Expression levels relative to β-actin were normalized by HuH7 CTRL. Data are shown as means ± SD (n = 3). *, **, and *** indicate statistical significance with P < .05, P < .01 and P < .001, respectively, vs CTRL. † and ‡ indicate P < .05 and P < .01, respectively, vs HuH7 cells. N.S. indicates not significant.
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13. Supplementary Figure 4
Effect of BMP-9 on iron metabolism-related genes expression. (A) HJV, (B) TfR2, (C) HEPC expressions. HuH7 cells were treated with the indicated concentrations of BMP-9 for 24 h, followed by real-time RT-PCR analysis. Expression levels relative to β-actin were normalized by 0 μM Fe CTRL. Data are shown as means ± SD (n = 3). *, **, and *** indicate statistical significance with P < .05, P < .01 and P < .001, respectively, vs 0 ng/mL of BMP-9.
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14. Supplementary Figure 5
Serological examinations of mice fed retinoid diets. (A) Serum iron levels were determined by Nitroso-PSAP method (Quickauto-Neo Fe; Shino-Test Corp, Kanagawa, Japan) according to manufacturer's instructions. (B) Serum ferritin levels were determined by latex immunoagglutination assay (LA ferritin; Nittobo Medical Co, Ltd, Tokyo, Japan) according to the manufacturer's instructions. Blood was taken from the heart and incubated at 4°C overnight. The serum was recovered by centrifugation. Data are shown as means ± SD (n = 5). *P < .05, **P < .01, ***P < .001.
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15. Supplementary Figure 6
Western blot analysis for ferroportin (Fpn) in the livers at day 5 after iron overload. Actin is shown as an internal control.
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