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. 2024 Nov 13;13(1):39. doi: 10.1038/s41389-024-00540-3

Fig. 3. PSPH overexpression and mutation favor GBM stem cell clonogenicity and growth.

Fig. 3

A The GBM 1919 was stably transduced with GFP containing vectors to test PSPH overexpression effects (PSPHamp) and the V116I mutant form of PSPH (PSPHV116I), using GFP control cells (GFP Ctrl). B Immunoblot analysis of PSPH protein expression in the different 1919 isogenic models, i.e. Ctrl, PSPHamp and PSPHV116I in n ≥ 3 biological replicates and n = 4 technical repeats. One-way ANOVA with Dunnett’s multiple comparison test has been performed. C Heatmap showing the percentage (%) of 13C-glucose derived mass distribution of m + 5 and m + 6 purines and pyrimidines nucleotides in PSPHamp and PSPHV116I GBM 1919 cells compared to 1919 GFP control cells under basal high glucose conditions in three biological replicates for each cell model. Two-way ANOVA test has been performed comparing each nucleotide to the 1919 GFP Ctrl. D Alvetex 3D clonogenic assays of PSPHamp and PSPHV116I GBM 1919 cells. Each individual dot represents and independent experiment. Unpaired 2-tailed t-test has been performed. E Representative images and graph of the migration rate of 1919 GFP control cells and 1919 PSPHamp and PSPHV116I cells in low glucose media. The dotted line limits the area of scratch at time 0. Area under the curve (AUC) of n = 3 independent experiments with n ≥ 10 wells analyzed/experiment has been calculated and normalized to 1919 GFP control cells. One-way ANOVA with Dunnet’s multiple comparisons test has been performed. F 13C-glucose derived mass distribution of NAD and NADP synthesis in three biological replicates for each cell model. Two-way ANOVA with Dunnett’s multiple comparisons test has been performed. G Intracellular levels of NAD and NADP in the isogenic models. One-way ANOVA with Dunnett’s multiple comparisons test has been performed. H Mitochondrial ROS measured using Mitotracker Red CM-H2XRos in GBM isogenic models. One-way ANOVA with Tukey’s multiple comparisons test has been performed. I Mito Stress Test was performed in 1919 isogenic models, measuring the oxygen consumption rate (OCR) using XFe96 Seahorse technology. Proton leak OCR was determined in n = 4 biological replicates and n = 4 technical replicates in each experiment. One-way ANOVA with Dunnett’s multiple comparisons test has been performed. Statistical significance p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****).