Fig 1: SerpinB3 is necessary for glioblastoma(A) Graphical abstract of His-JAM-A pulldown and liquid chromatography-mass spectrometry (LC-MS) procedure.(B) Verification of LC-MS results by western blot. His-tagged JAM-A was overexpressed in T4121 cancer stem cells (CSCs), and protein was isolated and mixed with nickel beads. The bound fraction was subjected to immunoblotting with antibodies to SerpinB3 and JAM-A.(C) Immunofluorescent staining demonstrating co-expression of JAM-A and SerpinB3 in T387 PDX glioblastoma tumor model (scale bar, 10 µm).(D) JAM-A was knocked down in T4121 CSCs with 2 separate shRNA constructs, and SerpinB3 expression was measured. Actin was used as a loading control in this and all subsequent western blots.(E) T387 and T4121 CSCs expressing JAM-A KD2 shRNA or NT control were treated with cycloheximide, and SerpinB3 expression was measured at 6 and 12 h post-treatment.(F) Western blot demonstrating knockdown of SerpinB3 with each shRNA, KD1, and KD2.(G) Fold change in cell viability at day 7, normalized to day 0, in 3 PDX glioblastoma models. Cell viability measured with CellTiter-Glo Luminescent Cell Viability Assay (5 technical replicates per condition, per tumor model).(H and I) Kaplan-Meier curves depicting survival of mice with 20,000 T4121 or T387 tumor cells intracranially injected. Cells were transfected with either non-target (SHC002) or SerpinB3 (KD1 or KD2) shRNA, with n = 10 mice per group.p < 0.05 was considered statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001, as determined by 1-way ANOVA with Dunnett’s multiple comparisons test or log rank test for survival data. Error bars represent standard deviations.