1. Supplementary Methods
Antitumor effect of treatments
To evaluate tumor growth, tumor volume was determined 3 times per week using two perpendicular diameters; tumor volume was calculated as V = (length x width2)/2. Treatments started when tumors reached a volume of 250 ± 50 mm3 (V0). Mice were sacrificed when tumors reached three times their initial volume (3V0). For each mouse, the time between the treatment onset and the animal sacrifice was defined as the “survival time” (in days).
Tumor growth delay (TGD) was defined as the difference [T - C], where T and C are the median survival time for the treated group and the control group, respectively.
We also calculated the radiopotentiation ratio (RR), defined as TGDBTZ + RT / [TGDBTZ + TGDRT], allowing to compare the observed effect for the association and the theoretical additive effect of BTZ alone and RT alone.
Proliferation and apoptotic index determination
Proliferation and apoptosis indexes were determined by immunohistochemistry as previously described (17). Briefly, tumors were excised and immediately fixed in 10% (v/v) buffered formalin pH 7.4 for 72 h and paraffin-embedded. Serial 5 µm sections were cut through the center of the xenografts. After section dewaxing, antigen retrieval was carried out with a 2100 Proteogenix Retriever (Proteogenix) following the manufacturer’s instructions. Sections were stained either with a species-unspecific rabbit monoclonal antibody (clone SP6, Lab vision Corporation; diluted 1:100) to the cell cycle-associated antigen Ki-67, or with an anti-cleaved caspase-3 rabbit anti-serum (BD Biosciences; diluted 1:1000). Primary antibodies were applied overnight at 4°C. Detection of the tissue-bound primary antibodies was performed using the biotinylated antibody/streptavidin-peroxidase detection system. Bound peroxidase was identified using the Novared™ detection system.
To determine proliferation and apoptotic indexes, the percentage of positively labeled tumor cells for at least 1,000 counted tumor cells was recorded in independent areas of the main tumor masses which were free of necrosis.
Cell cycle distribution
Cell cycle distribution was determined by DNA content analysis after propidium iodide staining, as previously described (18). Briefly, frozen tumor samples were incubated with a solution of trypsin (0.03 mg/mL) and were dissociated using a Potter pestle. Samples were then treated with a solution of Ribonuclease A (0.1 mg/mL) and incubated with a solution of propidium iodide (0.4 mg/mL) for 30 min in the dark. Before flow cytometry analysis, samples were filtered on a 25µm nylon mesh. Analysis of cell cycle distribution was performed using a FACSCalibur flow cytometer equipped with CellQuest and ModFit LT softwares (Becton Dickinson).

2. Supplementary Figure S1
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0.5
1.0
1.5
2.0
2.5
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NF-kB subunits xpression levels
Non tumoral brain tissue
TCG3 tumor tissue
U87 tumor tissue
Figure S1: Constitutive DNA-binding activities of NF-κB p65 and p50 subunits in TCG3 and U87 xenografts. DNA-binding activities of p65 and p50 were determined by an ELISA-based assay in TCG3 and U87 xenografts, as compared to samples of human non tumoral brain tissue. Results are expressed as the mean  SD of at 5 tumor samples.

3. Quantitative Real Time RT-PCR
Supplementary Table S1
Quantitative Real Time RT-PCR
Total RNA was extracted using TRIzol reagent (Invitrogen) from mg frozen tumor samples and 1µg of total RNA was used for reverse transcription reaction using the iScript™ cDNA Synthesis kit (Bio-Rad) according to manufacturer’s instruction.
The primers used for the amplification reaction for each gene are listed in Table S1. Real time PCR amplification was performed on a LightCycler (version 2.0, Roche) using QuantiFast SYBR Green PCR® (Qiagen) in accordance with the manufacturer’s protocol. The following cycling parameters were used for the PCR: 95° for 5 min, followed by 40 cycles of 95°C for 10 s and 60°C for 30 s. A final heating step up to 95°C was performed to obtain melting curve. Amount of mRNA of all target genes are normalized with the amount of β actin mRNA (housekeeping gene).
Table S1: Real time PCR primers characteristics
Gene
Primer Fwd
Primer Rev
Amplicon size (bp)
Tm (°C)
IL-6
5’-CAATTCTGGATTCAATGAGGAGAC
5’-CTCGGCTTGTTCCTCACTACTC
120 80
β Actin
5’-ATGTGGCCGAGGACTTTGAAT
5’-AGTGGGGTGGCTTTTAGGATG
107 77

4. Supplementary Figure S2
Figure S2: Treatment effects on IL-6 mRNA levels. mRNA level was determined by qRT-PCR and normalized to β actin mRNA level. Columns represent the median ratio between NF-κB target genes and β actin relative to untreated xenografts.

5. * Proteasome chymotrypsine-like activity (% CTRL) NFS FS #
Supplementary Figure S3
TCG3
U87 50
100
150
200
CTRL RT
BTZ+RT
NFS FS * #
Proteasome chymotrypsine-like
activity (% CTRL)
Figure S3: Bortezomib influence on the proteasome chymotrypsine-like activity in irradiated TCG3 and U87 glioma xenografts. Proteasome chymotrypsin-like activity was measured as previously described (14, 25). Activity was assessed 2 h after the last irradiation of the first week of treatment. Values for treatment groups were normalized to the basal proteasome activity measured in the CTRL group, defined as the 100%. Results are expressed as the median of at least four tumors. NFS: “non fractionated” schedule; FS: “fractionated” schedule.