Journal: Cell reports

Article Title: Targeting glioblastoma signaling and metabolism with a re-purposed brain-penetrant drug

doi: 10.1016/j.celrep.2021.109957

Figure Lengend Snippet: (A) Fluoxetine sensitivity (area under the cell viability curve) of 3 non-cancer cell lines and 18 patient-derived GBM neurospheres, including 4 EGFRvIII -amplified lines. (B) Relative cell viability of EGFRvIII -amplified GBM neurosphere lines with SMPD1 or non-targeting shRNAs (n = 4). (C) SMPD1 enzymatic activity in GBM neurospheres with 24 h of fluoxetine treatment (n = 4). (D) Percentage of Annexin V-positive cells in normal human astrocytes (NHAs) and GBM neurospheres (n = 4). (E and F) Gene set enrichment analysis identifies differentially enriched or depleted transcripts in three GBM neurosphere cultures treated with fluoxetine versus DMSO. (G and H) Gene set enrichment analysis of differentially expressed genes in TCGA GBM clinical samples (HUG133A) with high or low SMPD1 expression. (I) Drug sensitivity correlation of fluoxetine, 4 EGFR inhibitors, and 4 other SSRI antidepressants in 40 glioma cell lines from the DepMap dataset. (J) EGFR signaling in indicated GBM cells. (K) EGFR signaling in GBM39 cells with 24-h treatments. (L) EGFR phosphorylation in U87EGFRvIII orthotopic xenograft tumors. (M) Viability of GBM39 cells expressed vector or a constitutively active AKT E17A-CA allele (n = 4). (N) SMPD1 enzymatic activity in GBM39 cells treated with DMSO or 5 μM fluoxetine (n = 4). (O) Schematic of sphingomyelin (d18:1/n16:0-d9) metabolomics assay. (P and Q) Abundance of sphingomyelin (d18:1/n16:0-d9) (P) and ceramide (d18:1/n16:0-d9) (Q) in U87EGFRvIII cells with indicated treatment (n = 4). (R and S) Lipidomics analysis of endogenous sphingomyelins in U87EGFRvIII cells with 24 h of treatment and with SMPD1 overexpression (n = 5). Relative abundance of representative sphingomyelins is plotted in (S). (T) LAMP1 staining of GBM cells. Scale bar, 20 μm. Data represent mean ± SD, except for mean ± SEM in (S). Two-tailed Pearson (I). Two-tailed Student’s t test (R). ANOVA with Tukey’s multiple comparisons test (A–D, M, N, P, Q, and S). *p < 0.05, **p < 0.01, ***p < 0.001. See also – .

Article Snippet: 24:0 SM (d18:1/24:0) , Avanti Polar Lipids , Cat#860592.

Techniques: Derivative Assay, Amplification, Activity Assay, Expressing, Plasmid Preparation, Over Expression, Staining, Two Tailed Test

Journal: Cell reports

Article Title: Targeting glioblastoma signaling and metabolism with a re-purposed brain-penetrant drug

doi: 10.1016/j.celrep.2021.109957

Figure Lengend Snippet: (A and B) Laurdan imaging analysis of membrane lipid order in U87EGFRvIII cells at baseline, after fluoxetine treatment, and with overexpression of an SMPD1 construct. Generalized polarization (GP) images indicate higher membrane order (red) and lower membrane order (blue). Scale bar, 20 μm. (C and D) Imaging and flow cytometry analysis (n = 3) of cell surface EGFR in GBM cells. Scale bar, 10 μm. (E) EGFRvIII and marker proteins in the membrane fractions of GBM39 cells. Calnexin is a marker for non-lipid rafts fractions, while Gα(q) and Flotillin-1 are markers of lipid rafts. (F) Percentage of indicated protein levels in fraction 1, the lipid rafts fraction, which is absent with non-lipid rafts marker Calnexin and present with lipid raft markers Gα(q) and Flotillin-1. Data were normalized to total protein levels of all six fractions (n = 3). (G) Internalized EGFR of GBM39 cells by flow cytometry (n = 4). (H) EGFR signaling in GBM39 cells treated with sphingomyelins or vehicle. (I) EGFR staining in GBM39 cells with SM d18:1/n16:0 treatment. Scale bar, 10 μm. (J) Schematic model of the fluoxetine-SMPD1 axis in regulating sphingomyelin metabolism and oncogenic receptor signaling of GBM cells. Data represent mean ± SD. Two-tailed Student’s t test (D, F, and I). ANOVA with Tukey’s multiple comparisons test (G). *p < 0.05, ***p < 0.001. See also .

Article Snippet: 24:0 SM (d18:1/24:0) , Avanti Polar Lipids , Cat#860592.

Techniques: Imaging, Over Expression, Construct, Flow Cytometry, Marker, Staining, Two Tailed Test

Journal: Cell reports

Article Title: Targeting glioblastoma signaling and metabolism with a re-purposed brain-penetrant drug

doi: 10.1016/j.celrep.2021.109957

Figure Lengend Snippet:

Article Snippet: 24:0 SM (d18:1/24:0) , Avanti Polar Lipids , Cat#860592.

Techniques: Recombinant, Transfection, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Activity Assay, Plasmid Preparation, shRNA, Sequencing, Software