Additional Supporting Information may be found in the online version of this article.


Figure S1: Ncx1 is the predominant Ncx isoform expressed by mouse islet cells. (A) RT-PCR analysis of Ncx1, Ncx2 and Ncx3 expression. Isolated mouse islets (1000) were analyzed for mRNA levels by quantitative real-time PCR. Brain tissues and Gapdh were used as controls. (B) Fluorescent-micrographs of islets. Islets from wild-type C57BL/6 mouse pancreas were examined. The frozen sections were stained with DAPI (left column), anti-insulin (second column) and anti-NCXs (third column) (Suppl ref 1). The merged images are shown in the fourth column. Original magnification: 200×. Bars represent 50 μm.

Figure S2: NCX on MIN6 insulinoma cells functions during hypoxia/reoxygenation and is suppressed by a specific inhibitor, SEA0400. (A) [Ca2+]i elevation induced by hypoxia/reoxygenation in MIN6 cells is suppressed by a specific NCX inhibitor SEA0400. Typical traces (left panel) and average data (right panel) of Ca2+ signals when MIN6 cells expressing the Ca-indicator GCaMP2 were exposed to 5% CO2/95% air after culturing under hypoxic conditions for 6 h. The cells were treated with SEA0400 (10 μM) or vehicle. Each column and bar represent the mean ± SD of individual cells (n = 6–7). The difference between the two groups was statistically significant (*p < 0.001). (B) Oxygen tension in the medium during culture in hypoxia. Culture dishes were placed inside the chamber maintained at 37°C and gassed with 1% O2, 5% CO2 and 94% N2 and oxygen tension of the medium was measured continuously for more than 12 h using polarography (Unique Medical).

Figure S3: Use of Ncx1+/− islets to examine the role of NCX1 in in vitro hypoxic death and in vivo islet cell death after transplantation. (A) Dispersed cultured single cells prepared from Ncx1+/+ or Ncx1+/− MIP-GFP+ islets were used for the experiments. Typical traces (left panel) and average data (right panel) of Ca2+ signals when Ncx1+/− MIP-GFP+ islets were exposed to 5% CO2/95% air after hypoxic conditions for 6 h are shown. Each column and bar represents the mean ± SE at the time indicated. The differences were statistically significant compared with the corresponding controls (*p < 0.01, n = 3 and **p < 0.05, n = 3). (B) Fluorescent-micrographs of WT and Ncx1+/− islets cultured for 6 h in normoxia or in hypoxia as indicated. Cultured islets were stained with HO342 and PI. Representative micrographs of WT and Ncx1+/− islets are shown. Original magnification, 200×. Bars indicate 100 μm. (C) Nonfasting plasma glucose levels of STZ-induced diabetic mice transplanted with 200 syngeneic WT or Ncx1+/− islets. Lines represent individual animals. (D) Serum HMGB1 concentrations of mice receiving WT or Ncx1+/− islets at 6 h after transplantation. The difference was statistically significant (*p = <0.01, n = 3 in each group). (E) Flow cytometry profiles of MNC from the livers of mice receiving 200 WT or Ncx1+/− islets at 6 h after transplantation into the liver. Representative data of two experiments are shown.

Figure S4: The amount of insulin released from wild-type islets in vitro in response to glucose was not affected by treatment with SEA0400. Isolated islets were cultured overnight and used for the in vitro experiments. Islets were maintained at 37°C in a CO2-incubator (95% air + 5% CO2) throughout the experiment. Islets were first preincubated with KRH buffer containing 50 mg/dL glucose supplemental with 0.2% BSA for 2 h. Then, islets were further incubated in the KRH buffer containing 50 (n = 4) or 300 mg/dL (n = 4) glucose for 1 hour. The amount of insulin released in the medium after the last incubation was measured by ELISA (Morinaga, Tokyo, Japan). The values are expressed as mean ± SD (n = 4).

Figure S5: In vitro pretreatment of donor islets with SEA0400 helps prevent early loss of islet grafts transplanted beneath the kidney capsule of diabetic recipient mice. Nonfasting plasma glucose levels of STZ-induced diabetic mice transplanted with 50 syngeneic WT islets treated with 10 μM SEA0400 or vehicle (DMSO) prior to transplantation are shown. Lines represent plasma glucose levels of individual mice. *The kidneys bearing islet grafts were removed at the day indicated.

Table S1: Donors and islet characteristics

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