Fig 2: Increased HDAC6 activity upon glutamine starvation supports GS stability by deacetylating GS. mRNA levels of A) HDAC6 and B) GLUL analyzed with qRT‐PCR upon knockdown of HDAC6 in U251 cells. C) IB analysis showing the effect of MG132 (20 µm, 4 h) on HDAC6 knockout U251 cells. D) IB analysis and quantification graph showing the effect of CHX (100 µm) on HDAC6 knockout U251 cells. E) IP analysis showing acetylation of GS in HDAC6 knockdown U251 cells. Cells were incubated with 20 µm MG132 for 4 h before making cell lysate. F) IP analysis showing acetylation of GS in HDAC6‐overexpressing U251 cells. G) IP analysis showing the interaction between HDAC6 and either WT or 2KR mutant of GS in U251 cells. H) IP analysis showing the ubiquitination of GS in HDAC6 knockdown U251 cells. I) IB analysis showing HDAC6, Ac‐tub, and GS under glutamine starvation in U251 and U87 cells. Cells were incubated in Gln ± media for 24 h. J) GLUL mRNA levels analyzed with qRT‐PCR under glutamine starvation in U251 and U87 cells. Cells were incubated in Gln ± media for 24 h. IB analysis showing HDAC6, Ac‐tub, and GS levels under glutamine starvation for various time points in K) U251 and L) U87 cells. Relative HDAC6 activity in glutamine‐depleted condition relative to that in glutamine‐replete condition in M) U251 and N) U87 cells. Cells were incubated in Gln ± media for 24 h. O) HDAC6 activity of U251 and U87 cells. Data are shown as mean ± SD. ns, not significant; n = 3.

Fig 3: USP9X removes K48‐linked polyubiquitination on K91 and K103 of GS. A) Ubiquitination assay showing GS ubiquitination in USP9X knockdown U251 cells. B) Ubiquitination assay showing GS ubiquitination in USP9X knockdown U251 cells, detected with primary antibodies recognizing K48‐ and K63‐linked ubiquitin. C) Schematic diagram of the potential ubiquitination sites of GS. D) Ubiquitination assay showing the ubiquitination of GS mutants in U251 cells. E) CHX chase assay and quantification graph showing the protein stability of GS‐WT, ‐K91R, and ‐K103R. F) IB analysis showing the GS mutants after treatment with 4 mm glutamine following 48 h of glutamine deprivation. G) Ubiquitination assay showing the ubiquitination of GS‐WT, ‐K91R, and ‐K103R in USP9X knockdown U251 cells. H) Conservation of GS‐K91 and K103 across various organisms. I) IB analysis showing the effect of HDAC6 overexpression on USP9X knockdown U251 cells. J) IB analysis showing the effect of USP9X‐C overexpression on HDAC6 knockdown U251 cells. K) IP analysis showing the interaction between USP9X‐C and GS‐2KR in U251 cells. L) Ubiquitination assay showing GS‐2KR ubiquitination in U251 cells. Data are shown as mean ± SD, n = 3.

Fig 4: HDAC6 and USP9X positively correlate with GS and poor prognosis in GBM. Representative images and the number of stained samples from IHC staining of A) HDAC6 and B) USP9X in glioma and normal tissues. Data were obtained from the Human Protein Atlas database. IHC staining of C) HDAC6 and D) USP9X in TMA sections of GBM and normal tissues. E) Representative images of HDAC6, USP9X, and GS IHC staining in the GBM TMA sections. Scale bar, 20 µm. F–I) Correlation analysis between HDAC6, USP9X, and GS in the GBM TMA sections (n = 70). J,K) Kaplan–Meier survival analysis of primary GBM samples from the GEO dataset (GSE42669) stratified by HDAC6 and USP9X expression for overall survival (OS) and progression‐free survival (PFS). L) A Schematic diagram of HDAC6 and USP9X‐mediated GS regulation in GBM. Data are shown as mean ± SD.

Fig 5: Ablation of USP9X and HDAC6 impairs de novo nucleotide synthesis. A) Schematic illustration of glutamine metabolism. B) IB analysis showing that GS, HDAC6, and USP9X are knockdown in U251 cells. Relative levels of C) 15N2‐NH4Cl‐derived Gln, D) Glu, E) IMP, and F) UMP normalized to control NT cells. Cells were glutamine‐depleted for 12 h before labeling and incubated in glutamine‐free media containing 6 mm dmKG and 0.8 mm 15N2‐NH4Cl for 24 h. MRM transition of metabolites is summarized in Table S2 (Supporting Information). Colony formation assays in G) HDAC6 knockdown and H) USP9X knockdown U251 cells. Cells were incubated in Gln ± media supplemented with a combination of A, G, C, T, and U (0.2 mm each). I) Cell viability analysis of HDAC6 knockdown and USP9X knockdown U251 cells. Cells were incubated in Gln ± media supplemented with 0.2 mm each A, G, C, T, and U for 72 h, as indicated. Data are shown as mean ± SD; n = 3.