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. Author manuscript; available in PMC: 2021 Feb 20.
Published in final edited form as: Cell Chem Biol. 2019 Nov 13;27(2):197–205.e6. doi: 10.1016/j.chembiol.2019.10.012

Figure 3 |. OSU-03012 and TAK-632 inhibit DHODH and activate the DNA replication stress response pathway.

Figure 3 |

(A) Schematic of UMP biosynthesis via the de novo and salvage pathways. (B) Propidium iodide cell cycle analysis of MIAPACA2 PDAC cells treated ±5 μM TAK-632 or ±5 μM OSU-03012 and supplemented with 50 μM orotate (OA) or 10 μM rU (N.S.: no supplement). Insert indicates % S-phase cells. (C) Summary of fold changes in S-phase cells from B (mean±SD; n=2; one-way ANOVA corrected for multiple comparisons by Bonferroni adjustment, ns: not significant; * P<0.05; ** P<0.01). (D) in vitro DHODH enzyme assay performed in the presence of OSU-03012 or TAK-632. (E) Correlation between DHODH inhibitor (1 μM NITD-982) and OSU-03012 (3.17 μM) or TAK-632 (3.17 μM) response across a panel of 25 PDAC cell lines determined using CTG following 72 h treatment. Response calculated as doubling time normalized proliferation inhibition. Pearson correlation coefficient is indicated. (F) Immunoblot analysis of MIAPACA2 cells treated ±1 μM PDK1 inhibitor GSK-2334470 (GSK) ±1 μM OSU-03012 (OSU) ±10 μM rU for 24 h. (G) Immunoblot analysis of MIAPACA2 cells treated ±10 μM RAF inhibitor LY3009120 (LY) ±10 μM TAK-632 (TAK) ±10 μM rU for 24 h. (H) Annexin V/PI flow cytometry analysis of MIAPACA2 PDAC cells treated ±1 μM OSU-03012 or 1 μM GSK-2334470 (GSK) ±500 nM VE-822 (ATRi) ±25 μM rU for 72 h (mean±SD; n=2; one-way ANOVA corrected for multiple comparisons by Bonferroni adjustment; ns: not significant; ** P<0.01; *** P<0.001).