Fig.Figure 7(Left) Kaplan-Meier curve of the probability of survival for 155 GBM patients with low or high QKI RNA level, using median cut-off, based on the TCGA RNAseq dataset. (Right) Differential expression analysis related to either MALT1 or QKI expression highlighted a lysosomal lumen GO function. Venn diagram of overlapping lysosomal enriched protein encoding genes from this comparison showed 7 shared genes, together with 9 and 10 specific genes for MALT1 and QKI expression, respectively. (Bottom) Correlation between MALT1 and QKI expression was analyzed using The Cancer Genome Atlas (TCGA, HG-U133A dataset) on the GlioVis platform (Bowman et al, 2007). Pearson correlation factor=-0.21, p-value=0.03. GSCs #1 and #9 protein lysates (input) were processed for immunoprecipitation (IP) using control immunoglobulins (Ig), anti-QKI or anti-BCL10 antibodies. Input and IP fractions were separated on SDS-PAGE and western-blots for MALT1, QKI and BCL10 antibodies were performed as specified. Total protein lysates (input) from GSC#9 treated with vehicle (-, DMSO) or MPZ (+, 20 μM, 1 hour) or with vehicle (-, H2O) or Z-VRPR-FMK (+, 75 μM, 4 hours), were processed for control immunoglobulins (Ig) or anti-QKI antibodies immunoprecipitation (IP). Western-blots were performed with indicated antibodies. Western-blots were performed with indicated antibodies. Confocal analysis of Lysotracker (green) or FLAG (red) in GSC#9 overexpressing either empty vector (mock) or FLAG-QKI. Scale bars: 10 μm. Nuclei (DAPI) are shown in blue. Confocal analysis of LAMP2 (green) or FLAG (red) in GSC#9 transfected with either empty vector (mock) or FLAG-QKI. Scale bars: 10 μm. Nuclei (DAPI) are shown in blue. Quantification of LAMP2 staining pixel intensity on GSC#9 transfected with mock and FLAG-QKI. Data are presented as the mean + s.e.m. on 3 independent experiments. Each dot represents one cell. n>15. Western-blot analysis of QKI, LAMP2 and LC3B in GSC#9 overexpressing either empty vector (mock) or FLAG-QKI. GAPDH served as a loading control. Confocal analysis of mTOR (green) or FLAG (red) in GSC#9 transfected with either empty vector (mock) or Flag-QKI. Nuclei (DAPI) are shown in blue. Scale bars: 10 μm. GSC#1 were transfected with either empty vector (mock) or FLAG-QKI. Total protein lysates were processed for western-blots against p-S6 and FLAG. Total S6 served as a loading control. Fraction of surviving cells over time in GSCs #1 and #9, transduced with empty vector (mock) or FLAG-QKI bi-cistronic GFP plasmids. Data are plotted as the percentage of GFP-positive cells at the day of the analysis (Dx), normalized to the starting point (Day 4 post-infection, D4). Data are representative of n=3. GSC#9 transfected with non-silencing RNA duplexes (sic) or QKI targeting siRNA duplexes (siQKI) were treated for 16 hours with vehicle (DMSO) or MPZ (10 μM). Total protein lysates were processed for western-blots against LAMP2, CTSD, QKI, and LC3B expression, as indicated. GAPDH served as a loading control. Confocal analysis of mTOR (green) and LAMP2 (red) in GSC#9 transfected with sic or siQKI, and treated for 16 hours with vehicle (DMSO) or MPZ (20 μM). Nuclei (DAPI) are shown in blue. Scale bars: 10 μm. Quantification of mTOR colocalization score with LAMP2 is shown. The Coloc2 plug-in from ImageJ was used to measure Mander's tM1 correlation factor in LAMP2-positive ROI, using Costes threshold regression. Data are presented as the mean + s.e.m. on 3 independent experiments. Each dot represents one cell. n>10. GSC#9 transfected with non-silencing RNA duplexes (sic) or QKI targeting siRNA duplexes (siQKI) were treated for 1 hour with vehicle (DMSO) or MPZ (20 μM). Total protein lysates were processed for western-blots against QKI and p-S6. TUBULIN and Total S6 served as loading controls. FACS analysis of EdU staining was performed on GSC#9 cells transfected with non-silencing RNA duplexes (sic, pink), QKI targeting siRNA duplexes (siQKI, light purple), MALT1 targeting siRNA duplexes (siMALT1, blue) or double transfected with siQKI and siMALT1 (purple). FACS analysis of propidium iodide (PI) staining in GSC#9 transfected with non-silencing RNA duplexes (sic), QKI targeting siRNA duplexes (siQKI), MALT1 targeting siRNA duplexes (siMALT1) or double transfected with siQKI and siMALT1 and analyzed 72 hours later. Percentage of PI-positive cells normalized to vehicle treated controls are presented as the mean + s.e.m. on 3 independent experiments.