SEARCH

SEARCH BY CITATION

Figure S1. (A-C) Fluorescence micrographs of Etd uptake for 10 min in astrocytes under control conditions or after 3 h hypoxia in 27 mM glucose followed by 1 or 3 h reoxygenation. (D-F) Astrocytes and microglia co-cultured under control conditions or after 3 h hypoxia in 27 mM glucose followed by 1 or 3 h reoxygenation. (G-I) Astrocytes and microglia co-cultured only with Aβ25-35 for 24 h and then exposed to 3 h hypoxia in 27 mM glucose followed by 1 or 3 h reoxygenation. Scale bar, 150 µm.

Figure S2. (A-I) Fluorescence micrographs of scrape loading/dye transfer with LY in astrocytes under control conditions or after 3 h hypoxia in 27 mM glucose followed by 1 or 3 h reoxygenation. (D-F) Astrocytes and microglia co-cultured under control conditions or after 3 h hypoxia in 27 mM glucose followed by 1 or 3 h reoxygenation. (G-I) Astrocytes and microglia co-cultured only with Aβ25-35 for 24 h and then exposed for 3 h hypoxia in 27 mM glucose followed by 1 or 3 h reoxygenation. Scale bar, 100 µm.

Figure S3. CM-Aβ pretreatment followed by hypoxia-reoxygenation increases neuritic beading and death of neurons in co-culture with astrocytes. Representative confocal micrographs of immunofluorescence of MAP-2 staining (green) and Etd uptake (red) by neurons (N) cultured without and with astrocytes (Ast). (A) Neurons under control conditions or after 3 h hypoxia followed by 1 or 24 h reoxygenation. (B) After 3 h hypoxia and 1 h reoxygenation neurons appear normal. (C) After 24 h reoxygenation many cells show beading of neurites, but there is no Etd uptake indicating that cell membranes are intact. (D-F) Neurons incubated for 24 h with CM-Aβ prior to 3 h hypoxia and reoxygenation show death comparable to that in control medium. (G-I) Mixed neuron-astrocyte cultures show no beading or Etd uptake when subject to hypoxia-reoxygenation as in A–C; astrocytes are protective. (J–L) After 24 h pretreatment with CM-Aβ, cocultures show marked neuronal death and Etd uptake at 1 h and 24 h reoxygenation. (M–O) Neuronal death in cocultures caused by CM-Aβ pretreatment is prevented by the hemichannel blocker, Gap26, applied during reoxygenation.

Figure S4. Inhibition of NMDA and P2X receptors reduce the increase in F-Jade positive neurons induced by activated astrocytes. Representative confocal micrographs of immunofluorescence of MAP-2 (red) and F-Jade (green) staining by neurons under control conditions (A), exposed to CM-Ast for 1 h alone (B) or pretreated with 200 µM suramin, 10 U apyrase and 20 µM CPP (C). Scale bar, 80 µm.

Figure S5. Breakdown products of ATP does not affect neuronal hemichannel activity. (A) Etd uptake ratio normalized to control (dashed line) in neuronal cultures exposed for 1 h to various concentrations of ADP (white circles) and adenosine (black circles). (B) Etd uptake normalized to control (dashed line) in neuronal cultures exposed for 1 h to 100 µM ATP (white bars). Etd uptake was completely inhibited by two P2X7 receptor blockers: 200 µM oATP or 200 µM BBG, but nor by 1 µM DPCPX, an A1 adenosine receptor antagonist. *** p < 0.001, compared to the respective treatment. Each value corresponds to mean ± S.E. of 3 independent experiments.

As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.

FilenameFormatSizeDescription
JNC_7210_sm_FigS1.tif5264KSupporting info item
JNC_7210_sm_FigS2.tif8727KSupporting info item
JNC_7210_sm_FigS3.tif10819KSupporting info item
JNC_7210_sm_FigS4.tif4039KSupporting info item
JNC_7210_sm_FigS5.tif712KSupporting info item
JNC_7210_sm_SupplementaryFigure-legends.doc35KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.