Figure 5(Left) Confocal analysis of LAMP2 (red) and LC3B (green) in GSC#9 treated for 4 and 16 hours with vehicle (DMSO) and MPZ (20 µM). Nuclei (DAPI) are shown in blue. Scale bars: 10 μm. (Right) Super-resolution imaging (SIM, Structured Illumination Microscopy) of LAMP2 (red) and LC3B (green) staining in GSC#9 treated for 16 hours with vehicle (DMSO) or MPZ (20 µM).Western-blot analysis of LC3B and P62 in total protein lysates from GSC#9 at 0, 2, 4, 6, and 16 hours post-MPZ treatment (20 µM). GAPDH served as a loading control.Western-blot analysis of LC3B in total protein lysates from GSCs #1 and #9 at 72 hours post-transfection with sic or siMALT1, and subsequently treated 4 hours with vehicle (DMSO) or chloroquine (CQ, 20 µM). Knockdown was verified by MALT1 blotting and GAPDH served as a loading control.Confocal analysis of LC3B (green) in GSC#9 treated for16 hours with vehicle (DMSO) and MPZ (20 µM) with or without chloroquine (CQ, 20 µM). Nuclei (DAPI) are shown in blue. Scale bars: 10 μm.GSC#9 were transfected with LC3B reporters (wild type WT or G120A mutant, which cannot be lipidated), treated 24 hours later with vehicle (DMSO) or MPZ (20 µM) for 6 more hours. Ratios of WT/mutant luciferase signals are presented as the mean + s.e.m of 3 independent experiments.Confocal analysis of P62 staining (red) in GSC#9 treated for 16 hours with vehicle (DMSO) or MPZ (20 μM). Alternatively, GSC#9 were either transfected with sic or siMALT1 (middle) or treated for 16 hours with H2O or Z-VRPR-FMK (75 µM) (bottom). Quantification of P62 staining pixel intensity on GSC#9 treated for 16 hours with vehicle (DMSO or H2O), MPZ (20 µM) or Z-VRPR-FMK (75 µM) or sic and siMALT1. Data are presented as the mean + s.e.m. on 3 independent experiments. Each dot represents one cell. n>30.Cell viability was measuring using Cell TiterGlo in GSCs #1 and #9 pre-treated for 1 hour with vehicle (DMSO) or QVD (20 μM), and treated for 72 hours more with the indicated doses of MPZ. Data were normalized to the vehicle-treated controls and are presented as the mean + s.e.m of 4 independent experiments.FACS analysis of propidium iodide (PI) staining in GSC#9 treated for 48 hours with vehicle (DMSO) or MPZ (15 μM) in combination with QVD (20 μM). (Left) Percentage of PI positive cells, normalized to vehicle-treated controls are presented as the mean + s.e.m. on 3 independent experiments. (Right) Histogram plots for representative experiment (DMSO in red and MPZ in blue).CSTD ELISA was performed on culture media from GSC#9 treated for 8 hours with vehicle (DMSO) or MPZ (20 µM). Alternatively, cells were transfected with sic or siMALT1 and analyzed 72 hours later. Data are presented as the mean + s.e.m of 3 independent experiments.(Left) Cell viability was measured using Cell TiterGlo luminescent assay in GSC#9 treated for 48 hours with vehicle (DMSO) or MPZ (10 μM), following a 30 minute-pre-treatment with the following drugs: Bafilomycin A1 (Baf, 100 nM), Pepstatin A (Pep, 1 μg/mL), or CTS inhibitor 1 (Ctsi, 1 μM). Data were normalized to the vehicle-treated controls and are presented as the mean + s.e.m of 3 independent experiments in triplicate, stars refer to comparison to Vehicle + MPZ group (blue squares). (Right) FACS analysis of propidium iodide (PI) staining in GSC#9 treated for 48 hours with vehicle (DMSO) or MPZ (15 μM) in combination with Baf, Pep and Ctsi. Percentage of PI positive cells normalized to vehicle-treated controls are presented as the mean + s.e.m. on 3 independent experiments.