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. 2024 Jun 15;15:5115. doi: 10.1038/s41467-024-49298-7

Fig. 2. Validation of NF2/G6PD synthetic lethality.

Fig. 2

A–C sgRNA-mediated depletion of G6PD reduces the viability of NF2-KO but not NF2-WT cells. A Relative cell number 4 days after seeding (7 days postinfection), assessed by CellTiter-Glo. B Cell toxicity analyzed with CellTox, normalized to cell number. C Immunoblot to assess G6PD knock-out efficiency. Bars = mean ± SD. Significance by two-way ANOVA and Sidak’s multiple comparisons test. n = 5 (A) or 3 (B) independent experiments in biological quadruplicates. D Pharmacological inhibition of G6PD reduces the viability of NF2-KO but not NF2-WT Schwann cells. Bars = mean ± SD. Significance by one-way ANOVA and Dunnett’s multiple comparisons test. n = 5 biological replicates. E–F Pharmacological inhibition of G6PD strongly reduces viability of NF2-KO cells, as seen if the cells are passaged 3 times in the presence or absence of G6PDi-1. Passaging prevents wild-type cells from reaching confluence and no longer proliferating. Cells stained with crystal violet. E Representative image. F Significance by one-way ANOVA and Dunnett’s multiple comparisons test. n = 3 biological replicates. Bars = mean ± SD. G NF2-KO Schwann cells form tumors when implanted subcutaneously in NOD SCID gamma mice. Top panel: the macroscopic image of the excised tumor. Middle & bottom panels: hematoxylin and eosin stained tumor at two different magnifications. Representative of 10 tumors. H–I G6PD knockdown with shRNAs impairs the viability of NF2 KO cells. H Immunoblot control of G6PD knockdown. I Relative cell number 4 days after induction of control or G6PD shRNA, each compared to non-induced controls (DMSO). Bars = mean ± SD. Significance by one-way ANOVA and Dunnett’s multiple comparisons test. n = 4 biological replicates. J–K G6PD knockdown reduces the growth of NF2-KO Schwann cell tumor xenografts. Tumor volumes as a function of time of NSG mice injected subcutaneously and treated +/- Dox for 25 weeks. Renilla Luciferase (RLuc) shRNA = negative control. Animals were randomized into groups. Graphs show mean volumes; error bars=SEM. Significance by multiple unpaired t-test comparison analysis. L Inhibition of G6PD does not kill NF2-/+ heterozygous Schwann cells. Significance by one-way ANOVA and Dunnett’s multiple comparisons test. n = 3 biological replicates. Bars = mean ± SD Source data are provided as a Source Data file.