MOESM1 of 64Cu-ATSM/64Cu-Cl2 and their relationship to hypoxia in glioblastoma: a preclinical study

Additional file 1: Figure S1. Schematic representation of the protocol used for in vivo experiments. Figure S2. Evaluation of 64Cu-ATSM activity in the brain and the plasma. (A) Time activity curve (TAC) measured for 24 hours after the radiotracer injection in the tumor, peritumoral area and healthy brain. (B) Superposition of TAC in the blood and in the tumor quantified during 2 hours after 64Cu-ATSM injection. Figure S3. In vitro uptake of 64Cu-ATSM and 64Cu-Cl2 in tumor cells according the time incubation with radiotracer. Quantification of cell retention of 64Cu-ATSM (A) and 64Cu-Cl2 (B) in normoxic (21% O2) or hypoxic (0.5% and 0.2% O2) conditions after 1 hour or 4 hours of incubation of the tumor cells with the radiotracer brought into the culture medium. Mean ± SD, n = 3 different cell cultures per condition. Tukey’s HSD test after significant two-ways ANOVA (oxygen and time factors) was used: no significant difference was obtained. Figure S4. Spatial distribution of 64Cu-ATSM uptake in autoradiography at 3 hours or 24 hours after radiotracer injection. Figure S5. Protein expression of copper transporters, CTR1 and DMT1, in transient hypoxia. Cells were exposed to hypoxia (0.2% O2) during 24 hours and then reoxygenated (21%O2) during different times (6, 24 or 24 hours). Representative western-blot of DMT1 and CTR1 (A) and quantification of their protein expression (B). CAIX expression was used as positive hypoxic control. Mean ± SD, n = 3 different cell cultures per condition. Tukey’s HSD test after significant one-way ANOVA: * p < 0.05. Figure S6. Survival analyses of patients with glioblastoma according to the expression of copper transporters CTR1 and DMT1. Kaplan-Meier survival plot of glioblastoma patients were assessed according to the level of CTR1 or DMT1 gene expression from the REMBRANT database by using Betastasis online software (http://www.betastasis.com/, date of last access: 4.2.2012)

MOESM2 of 64Cu-ATSM/64Cu-Cl2 and their relationship to hypoxia in glioblastoma: a preclinical study

Additional file 2: Table S1. Details of primary and secondary antibodies used. IF=immunofluorescence, WB=western-blot. Table S2. Details of rat primers used for RT-qPCR analysis. Table S3. Quantification of immunostaining performed on brain slices with 64Cu-ATSM or 64Cu-Cl2 uptake in different areas R1, R2 and R3. Numbers in bold reflect immunolabeling greater than 75%

Endogenous erythropoietin as part of the cytokine network in the pathogenesis of experimental autoimmune encephalomyelitis

Erythropoietin (EPO) is of great interest as a therapy for many of the central nervous system (CNS) diseases and its administration is protective in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Endogenous EPO is induced by hypoxic/ischemic injury, but little is known about its expression in other CNS diseases. We report here that EPO expression in the spinal cord is induced in mouse models of chronic or relapsing-remitting EAE, and is prominently localized to motoneurons. We found a parallel increase of hypoxia-inducible transcription factor (HIF)-1 alpha, but not HIF-2 alpha, at the mRNA level, suggesting a possible role of non-hypoxic factors in EPO induction. EPO mRNA in the spinal cord was co-expressed with interferon (IFN)-gamma and tumor necrosis factor (TNF), and these cytokines inhibited EPO production in vitro in both neuronal and glial cells. Given the known inhibitory effect of EPO on neuroinflammation, our study indicates that EPO should be viewed as part of the inflammatory/anti-inflammatory network in MS