X. Luo, H. Zhao, L. Hennah, J. Ning, J. Liu, H. Tu, D. Ma

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Impact of isoflurane on malignant capability of ovarian cancer in vitro‡ X. Luo, H. Zhao, L. Hennah, J. Ning, J. Liu, H. Tu, D. Ma British Journal of Anaesthesia Volume 114, Issue 5, Pages 831-839 (May 2015) DOI: 10.1093/bja/aeu408 Copyright © 2015 The Author(s) Terms and Conditions

Fig 1 (a) Representative image of a single western blot and (b) band density analysis of of IGF-1 protein expression in SK-OV3 cells after 2% isoflurane exposure (n=4) and (c) IGF-1R expression after 1–2% isoflurane exposure using flow cytometry at 24 h (n=4). (d) Cell number after 2% isoflurane exposure using MTT assay (n=10). (e) Representative images of immunofluorescent staining of Ki-67 (nuclei counterstained with DAPI, scale bar: 50 µm) and (f) percentage of Ki-67-positive SK-OV3 cells after 2% isoflurane exposure (n=8). Data are expressed as mean (sd). *P<0.05, **P<0.01 and ***P<0.001. N2, nitrogen; NC, naïve control; Iso, isoflurane; MFI, mean fluorescence intensity. British Journal of Anaesthesia 2015 114, 831-839DOI: (10.1093/bja/aeu408) Copyright © 2015 The Author(s) Terms and Conditions

Fig 2 Representative images of (a) cyclin D and (c) cyclin E immunofluorescent staining in SK-OV3 cells at 4 and 24 h after 2% isofluorane exposure (nuclei counterstained with DAPI, scale bar: 50 µm). Fluorescence intensity of (b) cyclin D and (d) cyclin E (n=8). (e) Cell cycle analysis (percentage of cells in S phase) 24 h after exposure to 1–2% isoflurane (n=4). Data are expressed as mean (sd). *P<0.05 and ***P<0.001. N2, nitrogen; NC, naïve control; Iso, isoflurane. British Journal of Anaesthesia 2015 114, 831-839DOI: (10.1093/bja/aeu408) Copyright © 2015 The Author(s) Terms and Conditions

Fig 3 Representative image of immunofluorescent staining of (a) angiopoietin and (c) VEGF (nuclei counterstained with DAPI, scale bar: 50 µm) at 4 and 24 h after isoflurane exposure. Fluorescence intensity of (b) angiopoietin and (d) VEGF (n =8). (e) Tube formation in HUVEC cells after exposure to conditioned medium from SK-OV3 cells (CM) challenge for 24 h. (f) Number per x4 field of formed vascular tubes at 24 h after CM challenge (n=4). Data are expressed as mean (sd). *P<0.05, **P<0.01 and ***P<0.001. N2, nitrogen; NC, naïve control; Iso, isoflurane. British Journal of Anaesthesia 2015 114, 831-839DOI: (10.1093/bja/aeu408) Copyright © 2015 The Author(s) Terms and Conditions

Fig 4 (a) Representative image of SK-OV3 cell migration at 24 h and 48 h after isoflurane exposure using scratch assay. (b) Percentage healing (gap closure) after isoflurane exposure (n=8). Concentration of MMP-2 (c) and MMP-9 (d) in culture medium from SK-OV3 cells at 24 or 48 h after isoflurane exposure (n=4). Data are expressed as mean (sd). *P<0.05, **P<0.01 and ***P<0.001. N2, nitrogen; NC, naïve control; Iso, isoflurane. British Journal of Anaesthesia 2015 114, 831-839DOI: (10.1093/bja/aeu408) Copyright © 2015 The Author(s) Terms and Conditions

Fig 5 (a) Representative image of immunostaining of Ki-67 (nuclei counterstained with DAPI) and (b) percentage of Ki-67+ SK-OV3 cells 24 h after isoflurane exposure. (c) Percentage of cells in S phase, 24 h after isoflurane exposure. Representative images of immunostaining of (d) angiopoietin and (e) VEGF (nuclei counterstained with DAPI) and fluorescence intensity of (f) angiopoietin and (g) VEGF in SK-OV3 cells 24 h after αIR3-isoflurane treatment. Data are expressed as mean (sd) (n=4). *P<0.05, **P<0.01 and ***P<0.001. NC, naïve control; Iso, isoflurane. Scale bars: 50 µm. British Journal of Anaesthesia 2015 114, 831-839DOI: (10.1093/bja/aeu408) Copyright © 2015 The Author(s) Terms and Conditions

Fig 6 (a) Representative image of immunostaining of Ki-67 (nuclei counterstained with DAPI) and (b) percentage of Ki-67+ SK-OV3 cells 24 h after isoflurane exposure. (c) Percentage of cells in S phase, at 24 h after gas exposure. Representative images of immunostaining of (d) angiopoietin and (e) VEGF (nuclei counterstained with DAPI) and fluorescence intensity of (f) angiopoietin and (g) VEGF in SK-OV3 cells 24 h after siRNA-isoflurane treatment. Data are expressed as mean (sd) (n=4). *P<0.05 and **P<0.01. NC, naïve control; Iso, isoflurane; SS, scrambled siRNA; IS, IGF-1R siRNA. Scale bars: 50 µm. British Journal of Anaesthesia 2015 114, 831-839DOI: (10.1093/bja/aeu408) Copyright © 2015 The Author(s) Terms and Conditions