<p>Supplementary materials and methods. Detailed bioinformatics analysis methods (softwares commands and parameters) used to analyse in silicon TCGA and OKG exam sequencing data.</p>
<p>Supplementary figures and tables for bioinformatic analyses. Fig S1 TCGA bioinformatics analysis workflow chart Fig S2 alignment plots of quality score accuracy Fig S3 Screenshot of region of interest on Chr 19 indicating genotype information on KIR2DS4 variant call Table SI Characteristics of the patients and control population used in this study Table SII Clinical data of 300 GBM patient obtained from The Cancer Genome Atlas (TCGA) project. Table SIII Description of the 2504 control population from 1000 Genome Project. Table SIV Antibodies used for Flow cytometry</p>
Glioblastoma (GBM) is invariably resistant to temozolomide (TMZ) chemotherapy. Inhibiting the proteasomal pathway is an emerging strategy to accumulate damaged proteins and inhibit their lysosomal degradation. We hypothesized that bortezomib (BTZ) might sensitize GBM cells to TMZ. We examined change in autophagic flux after drug treatments and in combination with pharmacological inhibitors or CRISPR cas9 knockout of autophagy-related genes -5 and -7 (ATG5 and ATG7, respectively). Autophagic flux was increased in temozolomide resistant GBM cells as indicated by diminished levels of the autophagy markers LC3A/B-II and p62(SQSTM1), increased localisation of LC3A/B-II with STX17, higher long-lived protein degradation and no induction of apoptosis. In contrast, BTZ treatment abrogated autophagic flux by accumulation of LC3A/B-II and p62(SQSTM1) positive autophagosomes that did not fuse with lysosomes and reduced degradation of long-lived proteins. BTZ synergistically enhanced TMZ efficacy by attenuating cell proliferation, increased DNA damage and apoptosis. CRISPR Cas ATG5 knockout reversed BTZ-induced autophagy blockade and rescued the GBM treated cells from death. We conclude that bortezomib abrogates temozolomide induced autophagy through ATG5 dependent pathway.
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