Skip to main content
. Author manuscript; available in PMC: 2021 Mar 13.
Published in final edited form as: ACS Infect Dis. 2020 Jan 15;6(3):489–502. doi: 10.1021/acsinfecdis.9b00416

Figure 5. DHODH-like inhibitors of mVP24-induced ARE promoter activity.

Figure 5.

(A) ARE/mVP24 HEK293T cells were distributed in a 384-well plate and treated with increasing concentrations of the indicated compounds in triplicate. Twenty-four hours post-treatment firefly luciferase activity was assessed (left axis, red circles). In parallel, HEK293T cells were plated in a 384-well plate and treated in triplicate with increasing concentrations of compounds. Twenty-four hours post-treatment, ATP content was assessed to determine viability (right axis, black squares). Error bars represent the standard deviation. Structures of compounds are indicated. (B) ARE/mVP24 HEK293T cells were plated in a 384-well plate and treated with increasing concentrations of compound and 1 mM of uridine or deoxycytidine as indicated. Twenty-four hours post-treatment, firefly luciferase activity was assessed. Error bars represent the standard deviation. (C) Expression of Flag-tagged mVP24 (red circles) and endogenous Nrf2 (teal squares) expression determined relative to DMSO control for ARE/mVP24 HEK293T cells treated with the indicated compounds at 5 and 1 μM for 24h. The dotted line represents the DMSO control, error bars represent the standard deviation and individual values for each of the triplicate are indicated. (D) Immunoprecipitation of Flag-tagged mVP24 in cells also expressing HA-tagged Keap1, 24 hours post-treatment with 5 μM of the indicated compounds. Western blots were performed for Flag and HA. (E) HEK293T cells were transfected with plasmids encoding an ARE firefly luciferase reporter, a constitutively expressed Renilla luciferase and the indicated mVP24 and p53 expression plasmids. Twenty-four hours post-transfection, luciferase activity was assessed. Expression of Flag-tagged mVP24 and p53 was determined by western blot. Error bars represent the standard deviation. (F) ARE/mVP24 HEK293T cells were transfected with a scramble siRNA (scr) or one of two p53 targeted siRNAs (p53#1, p53#2) and were treated with compounds at indicated concentrations. Luciferase activity was assessed for the samples in triplicate twenty-four hours post treatment and siRNA knockdown was confirmed by western blot for p53. Statistical significance was assessed using unpaired t test; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Error bars represent the standard deviation.