Fig 1: Identification of key genes associated with 5-aminolevulinic acid (5-ALA) fluorescence intensity in glioblastoma. (A) Results of RNA-sequencing of classified samples, which were analyzed according to differential expression along with the fluorescence intensity (fold change >2, p < 0.05, FDR < 0.01). Seventy-seven genes with positive correlation and 509 genes with negative correlation between the expression level and fluorescence intensity were identified. (B) Pathway analysis using gene set enrichment analysis (GSEA) resulted in 30 significantly enriched gene sets of (p < 0.01, FDR < 0.25). Among them, GLS2 (red arrows) was repeatedly found with a high enrichment score in the selected gene sets of metabolism and the neuronal system. (C) From the results of the RNA-seq FPKM values, GLS2 was highly expressed in areas of no fluorescence intensity, and the expression was decreased in areas of positive fluorescence in all samples. (D) The expression level of GLS2 in all samples, as measured by quantitative real-time PCR, confirmed the result of RNA-seq.
Fig 2: The effect of etoposide on MYCN+ and MYCN- TET21N cells. a The level of MYCN in TET21N cells without and with doxycycline treatment; b caspase-3-like activity in MYCN+ and MYCN- TET21N cells after 6 h and 24 h of etoposide treatment; c GLS1 and GLS2 expression in glutamine-supported and -deprived MYCN+ and MYCN- TET21N cells; d glutathione content in MYCN+ and MYCN- TET21N cells treated with etoposide for 6 h. Concentration of etoposide—34 µM; *p < 0.05, **p < 0.01
Fig 3: The S47 variant is impaired for transactivation of a subset of p53 target genes, including Gls2, Noxa (Pmaip1), and Sco2. (A) qRT–PCR analysis of p53 target genes in primary wild-type (Wt) and S47 MEFs treated with 10 µM cisplatin (CDDP) for 24 h. All values were normalized to a control gene (cyclophilin A). Data are averaged from three independent biological replicates. Error bars indicate standard deviation. (*) P < 0.05. (B) qRT–PCR analysis of the p53 target genes indicated in independent batches of primary MEFs from wild-type and S47 mice treated with 10 µM CDDP for 24 h. All values were normalized to a control gene (cyclophilin A). Data are averaged from three independent biological replicates. Error bars indicate standard deviation. (*) P < 0.05. (C) qRT–PCR analysis of the p53 target genes indicated in human LCLs that are homozygous for wild-type p53 or S47 treated with 10 µM CDDP for 24 h. All values were normalized to a control gene (cyclophilin A). Data are averaged from three independent biological replicates. Error bars indicate standard deviation. (*) P =0.05. SCO2 was not expressed in LCL cells, so these data are not depicted. (D) Western analysis for the proteins indicated in wild-type and S47 MEFs pretreated with 10 µM p38MAPK inhibitor SB203580 for 2 h followed by 10 µM cisplatin (CDDP) for 24 h. GAPDH served as the loading control. (E) qRT–PCR analysis of the cells in D for the p53 target genes indicated, normalized to control (cyclophilin A). The depicted data represent the average of three independent experiments. Error bars represent standard deviation. (*) P <0.05.
Fig 4: GLS2 was upregulated during ferroptosis. (A,B) ACHN cells were treated with various concentrations of erastin (12 h) or RSL3 (6 h) with or without Fer-1 (1 µM), Lip-1 (1 µM), HCQ (20 µM), NSA (1 µM), Z-VAD (20 µM). (C,D) Caki-1 cells were treated with various concentrations of erastin (12 h) or RSL3 (6 h) with or without Fer-1 (1 µM), Lip-1 (1 µM), HCQ (20 µM), NSA (1 µM), Z-VAD (20 µM). (E) The cellular morphology of AHCN and Caki-1 treated with erastin (10 µM, 12 h) or RSL3 (1 µM, 6 h) in the absence or presence of Lip-1 (1 µM). The scale bar represented 500 µm. (F) The mRNA expression of nine FPDEGs in ACHN treated with erastin (10 µM, 12 h) or RSL3 (1 µM, 6 h) in the absence or presence of Lip-1 (1 µM). (G) The mRNA expression of nine FPDEGs in Caki-1 treated with erastin (10 µM, 12 h) or RSL3 (1 µM, 6 h) in the absence or presence of Lip-1 (1 µM). (* p < 0.05, ** p < 0.01, *** p < 0.001).
Fig 5: The glutamate/glutamine cycle. High-affinity excitatory amino acid transporters (EAATs) of astrocytes primarily clear Glu in the synaptic cleft that is released from presynaptic neurons. GLT1 (glutamate transporter 1/EAAT2/SLC1A2) is the major regulator that transports Glu to astrocytes. In astrocytes, Glu is converted into Gln by glutamine synthetase (GS). Synthesized Gln is released from the astrocytes through sodium-coupled neutral amino acid transporters (SNAT3/SLC38A3 and SNAT5/SLC38A5). Released Gln is transported into neurons via SNAT1 and SNAT2 (SLC38A1 and SLC38A2) and converted back to Glu by phosphate-activated glutaminase (PAG or GLS1/2, mainly GLS2 in brain) for synaptic transmission of glutamatergic neurons. Glutamate is further transformed to ?-aminobutyric acid (GABA) by glutamic acid decarboxylases (GAD1 and GAD2) in the GABAergic neurons. Glu can be synthesized in the astrocytes from glucose via the tricarboxylic acid (TCA) cycle, followed by transamination of a-ketoglutarate (a-KG). Pyruvate carboxylase (PC) converts pyruvate to oxaloacetate that is converted to a-KG via the TCA cycle. Glutamate dehydrogenase (GDH) connects the TCA and Glu-Gln cycles by reversibly converting Glu into a-KG and vice versa. Expressional changes by chronic immobilization stress (CIS) and Gln supplementation are marked by red and green arrows, respectively.
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