Journal: Advanced Science

Article Title: Prickle4 Drives Microenvironmental Remodeling and Resistance to Parp Inhibition in IDH‐Mutant Glioma

doi: 10.1002/advs.202503866

Figure Lengend Snippet: Mutant IDH1 and hypoxia induce DNA damage synergistically in IHA. A) GSEA analyses of significantly enriched gene sets related to angiogenesis. Blue dotted box: pathways related to mitotic activities. Red dotted box: pathways related to vasculature regulation. B) Comparison of average expression of 26 genes in IDHwt and IDHmut tumors. C) Kaplan‐Meier analysis comparing overall survival in angiogenesis score‐high and ‐low patients. P values were determined by the log‐rank (Mantel‐Cox) test. D) Western blot analysis of HIF‐1α and EZH2 (loading control) under hypoxia (1% O 2 ). Experiments were repeated three times, and the average signal intensity was plotted on bar graphs as mean ± SEM. N = 3. E) mRNA transcript abundance of LDHA, PDK1, PGK1, SLC2A1, VEGFA, and CA9 (internal control) was determined by Real‐time PCR and plotted on bar graphs as mean ± SEM. N = 4. F) Relative intracellular 2‐HG level in IHA‐EV and IHA‐IDH1mut under hypoxic and non‐hypoxic conditions measured by LC‐MS. N = 3. G) Immunofluorescence staining of γ‐H 2 AX (green) in IHA‐EV and IHA‐IDH1mut under hypoxic and non‐hypoxic conditions. Blue: DAPI. Scale bar represents 10 µm. H). Ten independent fields were quantified, and the percentage of average γ‐H 2 AX foci in the nucleus was plotted. N = 10. I) Neutral Comet assays determining DNA breaks in IHA‐EV and IHA‐IDH1mut under hypoxic and non‐hypoxic conditions. Scale bar represents 200 µm. J). The length of comet tails was quantified and represented on the plot. N = 20. K) propidium iodide (PI) positivity of IHA‐EV and IHA‐IDH1mut, treated with veliparib (20 µM) and hypoxia (1% O 2 ) was determined by flow cytometry, quantified, and plotted. N = 4. L) Clonogenic survival assays of IHA‐EV and IHA‐IDH1mut cells under the indicated conditions. The colonies were quantified and represented on the bar graph showing the synergistic effect of veliparib and hypoxia, specifically in IHA‐IDH1mut cells. N = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant.

Article Snippet: For transcript quantification, real‐time RT‐PCR was performed as described(Andersen et al, 2004) with the following Taqman probes: Ldha (ThermoFisher, Hs01378790_g1), Pdk1 (Hs01561847_m1), Pgk1 (Hs00943178_g1), Slc2a1 (Hs00892681_m1), Vegfa (Hs00900055_m1) and Ca9 (Hs00154208_m1) as internal control; for mouse tumors, Kdm5a (Mm00524457_m1), Prickle4 (Mm04933143_m1), Ddx3y (Mm00465349_m1), Ccl24 (Mm00444700_g1) and Esr1 (Mm00433149_m1) as internal control.

Techniques: Mutagenesis, Comparison, Expressing, Western Blot, Control, Real-time Polymerase Chain Reaction, Liquid Chromatography with Mass Spectroscopy, Immunofluorescence, Staining, Flow Cytometry