Fig 2. Inhibition of the de novo pyrimidine biosynthesis pathway decreases proliferation of temozolomide-sensitive and -resistant glioblasomta cells.

(A) Relative proliferation of p14 ARF-/- human astrocytes, LN229, GBM9 and SF188 cells with increasing amounts of brequinar. Media with drugs was replaced every 2 days for 6 days. N = 3. (B) Relative proliferation of LN229, GBM9 and SF188 cells with or without brequinar and uridine. Media with drugs was replaced every 2 days for 6 days. N = 3–4. (C) Representation of the effects of temozolomide (TMZ) and the depletion of nucleotides causing DNA double-strand breaks and the phosphorylation of H2AX (γ-H2AX). (D, E) Relative proliferation of LN229 and GBM9 in the presence of TMZ, brequinar (Breq.) or brequinar + TMZ. Media with drugs was replaced the day after seeding, and cell proliferation was measured 4 days later. N = 3–4. (F, G) Western blot of LN229 and GBM9 for γ-H2AX and p53. Also see Supplementary S2F and S2G Fig. (H) Representation of the in vitro generation of SF188 TMZ-resistant cells. (I) Relative proliferation of SF188 TMZ-sensitive and -resistant cells with or without TMZ. (J) Relative proliferation of SF188 TMZ-sensitive or -resistant cells with or without TMZ, brequinar or brequinar + TMZ with or without uridine normalize to each DMSO condition. Also see Supplementary S2H Fig. Media with drugs was replaced the day after seeding, and cell proliferation was measured 4 days later. (K) Western blot of SF188 TMZ-sensitive or -resistant cells with or without TMZ, brequinar or brequinar + TMZ with or without uridine for γ-H2AX, p53 and, MGMT. Also see Supplementary S2I and S2J Fig. (L) Cell cycle analysis of LN229 treated with 0.1 μM brequinar, 2 μM ML390 in the presence or absence of 100 μM uridine, and 100 μM TMZ for 24 h. Media with drugs was replaced the day after seeding and cells harvested after 24 h. Also see Supplementary S2K Fig. This experiment was done three times with similar results. (M) Western blot of ARPE, LN229, SF188 and GBM9 for p53, p21 and cleaved caspase 3 after 72 h of treatment with 0.1 μM brequinar in the presence or absence of 100 μM uridine. (N) Western blot of ARPE, LN229, and GBM9 for p53, p21 and cleaved caspase 3 after 72 h of treatment with 2 μM ML390 in the presence or absence of uridine 100 μM. (O) Western blot of ARPE, LN229, SF188 and GBM9 for p53, p21 and cleaved caspase 3 after 48 h of treatment with 1 μM brequinar or 4 μM ML390. For all the Western blot experiments, media with drugs and metabolites were replaced the day after seeding and cells harvested at the indicated time points. Anti-phosphorylated H2AX antibody shows non-ubiquitinated (~ 15 KDa) and ubiquitinated (~ 25 KDa) γ-H2AX. * indicates p-values ≦0.05. Numerical values for each of the experiments represented are available in S2 Data.