Supplementary Figure 4: Requirement of IMPDH activity for cell proliferation and compensatory role of IMPDH1 in IMPDH2 KO cells.

a, The indicated IMPDH KO GBM cells were generated by the CRISPR/Cas9 system as in the method. Western blot shows the upregulation of IMPDH1 in IMPDH2 KO LN229 cells. Compensatory role of IMPDH1 to support cell proliferation of IMPDH2 KO in LN229 and A172, similar to U87MG cells. Cells were maintained with 100 μM guanosine-supplemented media, which was replaced to DMEM/10% dialyzed FBS media 24 h before the assay. Data are presented as mean+s.d. n=3 biologically independent samples. One-way ANOVA. n=1 experiment for Western blot analysis. b, c, VX-497, a direct IMPDH inhibitor³³, and Mizoribine, a prodrug IMPDH inhibitor, suppress GBM cell proliferations. Unlike MPA and VX-497, mizoribine requires intracellular adenylate kinase to become an active form³⁴. Data are presented as an average of 3 biologically independent samples (b) and mean+s.d. n=3 biologically independent samples (c). Unpaired two-sided Student’s t-test. d, HPRT1 KO U87MG, LN229 and A172 cells can proliferate comparably to parental cells. Western blot analysis verified the depletion of HPRT1 proteins in the transduced cells. The HPRT1 KO U87MG, LN229 and A172 cells were resistant to 6-TG, while the proliferation of their parental cells was significantly decreased above 10 μM 6-TG (upper right). Cells were treated with 6-TG at the indicated concentrations for 72 or 96 h. The HPRT1 KO U87MG, LN229 and A172 cells show comparable proliferation to their parental cells under normal condition (lower right). Proliferation was assessed at 72 or 96 h after plating cells. n=1 experiment for Western blot analysis. Data are presented as mean+s.d. n=6 (U87MG), n=3 (LN229, A172) biologically independent samples. Unpaired two-sided Student’s t-test.