H. Wang, Y. Dong, J. Zhang, Z. Xu, G. Wang, C. A. Swain, Y. Zhang, Z

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Isoflurane induces endoplasmic reticulum stress and caspase activation through ryanodine receptors H. Wang, Y. Dong, J. Zhang, Z. Xu, G. Wang, C.A. Swain, Y. Zhang, Z. Xie British Journal of Anaesthesia Volume 113, Issue 4, Pages 695-707 (October 2014) DOI: 10.1093/bja/aeu053 Copyright © 2014 The Author(s) Terms and Conditions

Fig 1 Isoflurane increases CHOP levels in the primary neurones. (a) Immunohistochemistry staining of CHOP (magnification 20 ×). (b) Immunohistochemistry staining of CHOP (magnification 60 ×). Column 1 is the image of CHOP (green), column 2 is the image of nuclei (blue), and column 3 is the merged image. Row a is the cells following the control condition and row b is the cells treated with 2% isoflurane for 6 h. (c) Quantification of the immunohistochemistry staining shows that the isoflurane treatment (green striped bar) increases CHOP levels compared with the control condition (blue bar). British Journal of Anaesthesia 2014 113, 695-707DOI: (10.1093/bja/aeu053) Copyright © 2014 The Author(s) Terms and Conditions

Fig 2 Isoflurane increases the levels of CHOP and caspase-12 in the primary neurones. (a) Treatment with 2% isoflurane for 6 h (lanes 4–6) increases CHOP levels when compared with the control condition (lanes 1–3) in the primary neurones. There is no significant difference in the amounts of β-actin in the control condition- or isoflurane-treated neurones. (b) Quantification of the western blot shows that isoflurane treatment (green striped bar) increases CHOP levels compared with the control condition (blue bar), normalized to β-actin levels. (c) Treatment with 2% isoflurane for 6 h (lanes 4–6) increases cleaved caspase-12 levels when compared with the control condition (lanes 1–3) in the primary neurones. There is no significant difference in the amounts of β-actin in the control condition- or isoflurane-treated neurones. (d) Quantification of the western blot shows that the isoflurane treatment (green striped bar) increases cleaved caspase-12 levels compared with the control condition (blue bar), normalized to β-actin levels. (e) Treatment with 2% isoflurane for 6 h (lanes 3 and 4) increased cleaved caspase-3 levels when compared with the control condition (lanes 1 and 2). There is no significant difference in the amounts of β-actin in the control condition- or isoflurane-treated neurons. (f) The quantification of western blot shows that the isoflurane treatment (green striped bar) induces caspase-3 activation when compared with control condition (blue bar). British Journal of Anaesthesia 2014 113, 695-707DOI: (10.1093/bja/aeu053) Copyright © 2014 The Author(s) Terms and Conditions

Fig 3 Treatment with 2% isoflurane for 3 h induces ER stress without caspase-3 activation in the primary neurones. (a) Treatment with 2% isoflurane for 3 h (lanes 4–6) increases CHOP levels when compared with the control condition (lanes 1–3) in the primary neurones. There is no significant difference in the amounts of β-actin in the control condition- or isoflurane-treated neurones. (b). Quantification of the western blot shows that the isoflurane treatment (green striped bar) increases CHOP levels compared with the control condition (blue bar), normalized to β-actin levels. (c) Treatment with 2% isoflurane for 3 h (lanes 4 and 6) increases cleaved caspase-12 levels when compared with the control condition (lanes 1–3) in the primary neurones. There is no significant difference in the amounts of β-actin in the control condition- or isoflurane-treated neurones. (d) Quantification of the western blot shows that the isoflurane treatment (green striped bar) increases the cleaved caspase-12 levels compared with the control condition (blue bar), normalized to β-actin levels. (e) Treatment with 2% isoflurane for 3 h (lanes 4–6) does not induce caspase-3 activation when compared with the control condition (lanes 1–3) in the primary neurones. (f) Quantification of the western blot shows that the isoflurane treatment (green striped bar) does not induce caspase-3 activation compared with the control condition (blue bar), normalized to β-actin levels. British Journal of Anaesthesia 2014 113, 695-707DOI: (10.1093/bja/aeu053) Copyright © 2014 The Author(s) Terms and Conditions

Fig 4 Treatments with 1% or 2% isoflurane for 1, 3, and 6 h on CHOP levels in primary neurones of mice. Treatment with 2% isoflurane for 1 h does not increase CHOP levels in the neurones (a and b). The treatments with 1% isoflurane for 1 (c and d), 3 (e and f), and 6 (g and h) h do not increase CHOP levels in the neurones. British Journal of Anaesthesia 2014 113, 695-707DOI: (10.1093/bja/aeu053) Copyright © 2014 The Author(s) Terms and Conditions

Fig 5 Treatments with 1% or 2% isoflurane for 1, 3, and 6 h on caspase-12 activation in primary neurones of mice. Treatment with 2% isoflurane for 1 h does not induce caspase-12 activation in the neurones (a and b). The treatments with 1% isoflurane for 1 (c and d), 3 (e and f), and 6 (g and h) h do not induce caspase-12 activation in the neurones. British Journal of Anaesthesia 2014 113, 695-707DOI: (10.1093/bja/aeu053) Copyright © 2014 The Author(s) Terms and Conditions

Fig 6 Treatments with 1% or 2% isoflurane for 1, 3, and 6 h on caspase-3 activation in primary neurones of mice. Treatment with 2% isoflurane for 1 h does not induce caspase-3 activation in the neurones (a and b). The treatments with 1% isoflurane for 1 (c and d), 3 (e and f), and 6 (g and h) h do not induce caspase-3 activation in the neurones. The treatments with 2% isoflurane for 1 h (a and b), 1% isoflurane for 3 h (e and f), and 1% isoflurane for 6 h (g and h) decrease the caspase-3 activation. British Journal of Anaesthesia 2014 113, 695-707DOI: (10.1093/bja/aeu053) Copyright © 2014 The Author(s) Terms and Conditions

Fig 7 Dantrolene attenuates the isoflurane-induced ER stress and caspase-3 activation in the primary neurones. (a) Treatment with 2% isoflurane plus DMSO for 6 h (lane 3) increases CHOP levels when compared with the control plus DMSO condition (lane 1) in the primary neurones. Treatment with isoflurane plus dantrolene (lane 4) leads to a reduction in the CHOP levels when compared with the treatment with isoflurane plus DMSO (lane 3). There is no significant difference in amounts of β-actin among the different groups. (b) Quantification of the western blot shows that isoflurane plus DMSO treatment (pink striped bar) increases CHOP levels when compared with the control plus DMSO condition (blue bar), normalized to β-actin levels, whereas dantrolene (orange striped bar) attenuates the isoflurane-induced increases in the CHOP levels, normalized to β-actin levels. (c) Treatment with 2% isoflurane plus DMSO for 6 h (lanes 7–9) increases the cleaved caspase-12 levels when compared with the control plus DMSO condition (lanes 1–3) in the primary neurones. Treatment with isoflurane plus dantrolene (lanes 10–12) leads to a lesser degree of cleaved caspase-12 levels compared with the treatment with isoflurane plus DMSO (lanes 7–9). There is no significant difference in amounts of β-actin among different groups. (d) Quantification of the western blot shows that isoflurane plus DMSO treatment (pink striped bar) increases the cleaved caspase-12 levels compared with the control plus DMSO condition (blue bar), normalized to β-actin levels, whereas dantrolene (orange striped bar) attenuates the activation of caspase-12 induced by isoflurane plus DMSO (pink striped bar), normalized to β-actin levels. (e) Treatment with 2% isoflurane plus DMSO for 6 h (lanes 7–9) induces caspase-3 activation when compared with the control plus DMSO condition (lanes 1–3) in the primary neurones. Treatment with isoflurane plus dantrolene (lanes 10–12) induces a lesser degree of caspase-3 activation when compared with the treatment with isoflurane plus DMSO (lanes 7–9). There is no significant difference in the amounts of β-actin among different groups. (f) Quantification of the western blot shows that isoflurane plus DMSO treatment (pink striped bar) induces caspase-3 activation compared with the control plus DMSO condition (blue bar), normalized to β-actin levels, whereas dantrolene (orange striped bar) attenuates the isoflurane-induced caspase-3 activation, normalized to β-actin levels. British Journal of Anaesthesia 2014 113, 695-707DOI: (10.1093/bja/aeu053) Copyright © 2014 The Author(s) Terms and Conditions