human brain tumor tissue samples whole human brain tissue lysates  (Novus Biologicals)

Journal: iScience

Article Title: Inhibition of the pseudokinase MLKL alters extracellular vesicle release and reduces tumor growth in glioblastoma.

doi: 10.1016/j.isci.2022.105118

Figure Lengend Snippet: Figure 1. The expression of the pseudokinase MLKL is associated with aggressive glioma (A) The Cancer Genome Atlas (TCGA RNAseq dataset) was interrogated via the GlioVis platform to analyze the probability of survival (log-rank p-value) in 156 GBM patients, stratified based on low vs high expression (median cut-off) of 69 EV-related genes. Probability of survival was significantly changed for nine of them (namely SYTL5, SYTL3, SNAP25, STX6, RAB11B, RAB27A, MLKL, AIP, and CHMP4A). (B) Fold changes in mRNA expression for the 9 genes selected from panel (a) in non-tumor vs GBM patients. (C) Box plots of MLKL mRNA expression in non-tumor, low-grade glioma (LGG grades II and III), and in GBM (grade IV), according to the mutation status of IDH1 (IDH1mut) and the GBM subtypes (classical CL, mesenchymal MES, neural NEU, and proneural PN), based on the TCGA RNAseq and Agilent4502A datasets. GBM status: non-tumor n = 4, GBM n = 156; Grades: II n = 226, III n = 244, IV n = 150; IDH: WT n = 339, mut n = 27; Subtypes: CL n = 86, MES n = 96, NEU n = 111, PN n = 238. (D) Kaplan-Meier curve of the probability of survival for n = 244 GBM patients with low vs high expression of MLKL, using optimal cut-off settings in the TCGA dataset. (E) Protein lysates from tumor and control (healthy) human brain tissue biopsies were analyzed by immunoblot for MLKL protein expression (arrow). The hashtag symbol indicates a non-specific signal. GAPDH serves as a loading control. (F) MLKL protein expression was assessed in three patient-derived Glioblastoma Stem-like Cells from mesenchymal (GSC1 and GSC4) and classical (GSC9) subtypes. GAPDH serves as a loading control. Immunoblots are representative of at least n = 3. t-test and ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, ns not- significant.

Article Snippet: P65 Santa Cruz Biotechnology Cat#sc-372; RRID: AB_632037 PARP Santa Cruz Biotechnology Cat#sc-8007; RRID: AB_628105 phospho-ATM Cell Signaling Technology Cat#5883; RRID: AB_10835213 phospho-Histone H3 Cell Signaling Technology Cat#3377; RRID: AB_1549592 phospho-IkBa Cell Signaling Technology Cat#9246; RRID: AB_2267145 phospho-MLKL Abcam Cat#187091; RRID: AB_2619685 phospho-p65 Cell Signaling Technology Cat#3033; RRID: AB_331284 RAB14 Santa Cruz Biotechnology Cat#sc-271401; RRID: AB_10610486 RAB27A Cell Signaling Technology Cat#95394; RRID: AB_2800247 RIPK1 BD Biosciences Cat#551042; RRID: AB_394015 RIPK3 Cell Signaling Technology Cat#13526; RRID: AB_2687467 SOX2 Merck Millipore Cat#AB5603; RRID: AB_2286686 STEAP3 Abcam Cat#ab151566 TUBULIN Santa Cruz Biotechnology Cat#sc-8035; RRID: AB_628408 Biological samples Patient-derived glioblastoma cells GSC1, GSC4, GSC9 Harford-Wright et al. (2017) N/A Human Brain (Normal) tissue lysate GeneTex Cat#GTX28771 Human Brain Whole Tissue Lysate (Adult Whole Tumor) Novus Biologicals Cat# NB820-59423 (Continued on next page) iScience 25, 105118, October 21, 2022 19

Techniques: Expressing, Mutagenesis, Control, Western Blot, Derivative Assay

Journal: iScience

Article Title: Inhibition of the pseudokinase MLKL alters extracellular vesicle release and reduces tumor growth in glioblastoma

doi: 10.1016/j.isci.2022.105118

Figure Lengend Snippet:

Article Snippet: Human Brain Whole Tissue Lysate (Adult Whole Tumor) , Novus Biologicals , Cat# NB820-59423.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, EdU Assay, Flow Cytometry, Imaging, Cell Counting, Luciferase, Viability Assay, Isolation