Volume 24, Issue 1, Pages 9-15 (March 2017)

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Volume 24, Issue 1, Pages 9-15 (March 2017) Role of GPx4 in human vascular endothelial cells, and the compensatory activity of brown rice on GPx4 ablation condition Osamu Sakai, Toshinori Yasuzawa, Yoshie Sumikawa, Takashi Ueta, Hirotaka Imai, Akiyoshi Sawabe, Shigeru Ueshima Pathophysiology Volume 24, Issue 1, Pages 9-15 (March 2017) DOI: 10.1016/j.pathophys.2016.11.002 Copyright © 2016 Elsevier B.V. Terms and Conditions

Fig. 1 Knockdown of GPx4 siRNA in HUVEC. (A) Knockdown efficiency evaluated by mRNA levels (n=4). (B) Knockdown efficiency evaluated by protein levels using immunoblot analysis. (C) Phase contrast morphology of HUVEC transfected with siRNA of scramble control and GPx4 24h after transfection. Scale bar, 50μm. Pathophysiology 2017 24, 9-15DOI: (10.1016/j.pathophys.2016.11.002) Copyright © 2016 Elsevier B.V. Terms and Conditions

Fig. 2 Determination of lipid peroxidation. (A) Detection of 4-HNE by fluorescence microscopy using 4-HNE antibodies. (B) The fluorescence intensities of 4-HNE were quantified using ImageJ (NIH) (n=4-5). ##P<0.01 relative to control siRNA group (Student’s t-test). **P<0.01 relative to GPx4 siRNA group (Dunnet-test). α-Toc=α-tocopherol. Pathophysiology 2017 24, 9-15DOI: (10.1016/j.pathophys.2016.11.002) Copyright © 2016 Elsevier B.V. Terms and Conditions

Fig. 3 LDH from HUVEC treated with control or GPx4 siRNA 4days after transfection. (A) α-tocopherol (α-Toc) and brown rice extract prevented the LDH release induced by GPx4 knockdown (n=4). ##P<0.01 relative to control siRNA group (Student’s t-test). **P<0.01 relative to GPx4 siRNA group (Dunnet-test). (B) Ferrostatin-1 prevented the LDH release induced by GPx4 knockdown (n=4). ##P<0.01 relative to control siRNA group (Student’s t-test). *P<0.05 relative to GPx4 siRNA group (Student’s t-test). Pathophysiology 2017 24, 9-15DOI: (10.1016/j.pathophys.2016.11.002) Copyright © 2016 Elsevier B.V. Terms and Conditions

Fig. 4 Proliferation of HUVEC treated with GPx4 siRNA. Proliferation was evaluated by WST-8 assay at 4days after transfection. (A) α-tocopherol (α-Toc) and brown rice extract ameliorated suppression of proliferation by GPx4 knockdown (n=4). ##P<0.01 relative to control siRNA group (Student’s t-test). **P<0.01 and *P<0.05 relative to GPx4 siRNA group (Dunnet-test). (B) Ferrostatin-1 ameliorated suppression of proliferation by GPx4 knockdown (n=4). ##P<0.01 relative to control siRNA group (Student’s t-test). **P<0.01 relative to GPx4 siRNA group (Student’s t-test). Pathophysiology 2017 24, 9-15DOI: (10.1016/j.pathophys.2016.11.002) Copyright © 2016 Elsevier B.V. Terms and Conditions

Fig. 5 The influence of GPx4 knockdown on apoptosis activation in HUVEC. Caspase-3 and PARP cleavage were detected by immunoblot analysis using specific antibodies for caspase-3 and PARP. Staurosporine (1μM) served as a positive control. Pathophysiology 2017 24, 9-15DOI: (10.1016/j.pathophys.2016.11.002) Copyright © 2016 Elsevier B.V. Terms and Conditions