Fig 2: Upregulation of IMPDH2 in human and murine GBM.a, Principal component analysis in ten clinically relevant glioma mouse models by Illumina cDNA microarray (upper). Black dots are control normal brain samples, red dots are glioma samples. The experiment contains the gene expression profiles from 13 normal and 59 glioma mice (n=72). Impdh2 expression at mRNA levels (lower left) and immunohistochemical (IHC) analysis of IMPDH2 in the Nestin-driven Pdgf-B in lnk4a-arf-/-;Ptenfl/fl mouse brain (lower right). Boxplots follow a Turkey style, in which lower and upper hinges correspond to the first and third quartiles. The IHC image is a representative one from 4 independent animals. Scale bar indicates 1000 µm.b, A schematic diagram of purine biosynthesis pathway.c, GBM tissue from the GBM PDX mouse expresses IMPDH2, while IMPDH1 protein was undetectable level. n=1 experiment.d, Analysis of four cohorts showed the increased IMPDH2 expression in human glioma specimens with different WHO grades (bottom). H&E staining of GBM and representative IHC for IMPDH1 and IMPDH2. Data are presented as mean±s.e.m. n=91 for Cohort#1, n=191 for Cohort#2, n=91 for Cohort#3, n=53 for Cohort#4. One-way ANOVA. The IHC image is the representative one from Cohort#4 (n=53). Scale bar indicates 300 µm.e, Kaplan-Meier survival curves shown for three cohorts of glioma patients on the basis of IDH mutational status (Cohort #1 (n=90), #3 (n=38)) and the relative strength of cytoplasmic IMPDH2 expression. Cohort #4 (n=32) is progression-free survival and the others are overall survival. Log-rank tests (two-sided) were used for the statistical analysis.

Fig 3: IMPDH2 reprograms GTP-metabolism in GBM.a, Biosynthetic rate of GTP, but not ATP, is decreased by pharmacological inhibition of IMPDH in U87MG cells. Isotopomer distribution (M+6) of GTP and ATP from [U-13C]glucose is shown in right. Data are presented as mean+s.d. n=3 biologically independent samples. Unpaired two-sided Student’s t-test.b, GTP levels were decreased by 4 h of MPA treatment in U87MG cells. n=1 experiment.c, Pharmacological inhibition of IMPDH activity leads to acute decrease of GTP concentration, but not ATP in GBM cells. The indicated cells were treated with 10 µM MPA for 4 h and GTP and ATP concentrations were quantified by HPLC for U251 cells and CE-MS for LN229 cells. Data are presented as mean+s.d. n=3 biologically independent samples. Unpaired two-sided Student’s t-test.d, The indicated IMPDH KO U87MG cells were generated by the CRISPR/Cas9 system as in the Method. Western blot shows the upregulation of IMPDH1 in IMPDH2 KO U87MG and LN229 cells. Cells were maintained with 100 µM guanosine supplemented media, which was replaced to DMEM/10% dialyzed FBS media 24 h before the assay. GTP levels were quantified by HPLC. Data are presented as mean+s.d. n=3 biologically independent samples. One-way ANOVA. Western blot analysis was performed at least twice.e, IMPDH2 KO and IMPDH DKO U87MG decreased biosynthetic rate of GTP. The assay was performed as in (a). Data are presented as mean+s.d. n=3 biologically independent samples. One-way ANOVA.

Fig 4: Immunohistochemical staining of (A) IMPDH2 and (B) HPRT in osteosarcoma tissues (?400). (C) Distribution of IMPDH2 and HPRT levels in osteosarcoma cohort (N = 127). H&E; hematoxylin and eosin.

Fig 5: IMPDH2 is a potential target of BTBD9 in sleep regulation. (A) Scatter chart showing the correlation analysis result of BTBD9 expression and IMPDH2 expression in distinct brain area (cortex, midbrain, hippocampal, basal ganglia, hypothalamus, and amygdala). (B) Circular Manhattan plot GWAS showing the association of SNPs 2MB up- and downstream of IMPDH2 with sleep traits (N1/TST, N2/TST, N3/TST, WK/SPT, TST, and SE from innermost to outside). (C) Manhattan plot showing the association of SNPs related to N1/TST (p < 10–3).