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. 2020 Jan 1;10(3):998–1015. doi: 10.7150/thno.40076

Figure 7.

Figure 7

ZDHHC17-MAP2K4 Signaling Module is Required for GBM Cell Neurosphere Formation. (A-C) Flow cytometry and quantitation of SOX2 (stem cell marker) (A, B) and western blot of SOX2, CD133, and Nestin using β-actin as a loading control (C) in SW1088 cells transfected with ZDHHC17 plasmid or control, further transfected with MAP2K4 dsRNA, or genistein (2.5 μM)-treated. Data represent the means ± SD from three separate experiments (***p < 0.001, unpaired t-test). (D) Self-renewal of glioma stem cells (GSCs) originating from U118MG cells transfected with ZDHHC17 or MAP2K4 dsRNA, or genistein (2.5 μM) or 2-bromopalmitate (palmitoylation inhibitor; 200 μM)-treated, by neurosphere formation assay. Neurosphere numbers from dsRNA-transduced or inhibitor-treated ZDHHC17-expressing GSCs were calculated. Data represent the means ± SD from three separate experiments (ns, not significant; ***p < 0.001, unpaired t-test). (E) Neurosphere capacity by serial dilution assay of GSCs from U118MG cells transfected with ZDHHC17 or MAP2K4 dsRNA, or genistein (2.5 μM) or 2-bromopalmitate (200 μM)-treated. Data represent the means ± SD from three separate experiments (ns, not significant; ***p < 0.001, unpaired t-test). (F) Colonies formed by 200 viable GSCs from U118MG cells transfected with MAP2K4 dsRNA or ZDHHC17, or genistein (2.5 μM) or 2-bromopalmitate (200 μM)-treated. Data represent the means ± SD from three separate experiments (ns, not significant; ***p < 0.001, unpaired t-test). (G) Representative photon flux images from BALB/c mouse brains implanted with luciferase-expressing GSCs from U118MG cells transfected with control (left) or ZDHHC17 shRNA (right), then further genistein (50 mg kg-1 two days-1) or 2-bromopalmitate (100 mg kg-1 day-1)-treated. (H) Bioluminescence signal intensity in intracranial tumors between control (left) or ZDHHC17 shRNA (right) U118MG GSCs with or without 2-bromopalmitate or genistein (n = 5). Data represent the means ± SD (ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001, unpaired t-test). (I) Kaplan-Meier survival curve of animal survival following control or ZDHHC17 shRNA U118MG GSC cell injection with or without genistein or 2-bromopalmitate.