Research ArticleCELL BIOLOGY

High-throughput small molecule screening reveals Nrf2-dependent and -independent pathways of cellular stress resistance

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Science Advances  02 Oct 2020:
Vol. 6, no. 40, eaaz7628
DOI: 10.1126/sciadv.aaz7628
  • Fig. 1 Primary screen for protection of mouse cells from PQ lethality.

    (A) Example of a single experiment screening 640 compounds. No PQ wells (n = 32) received neither PQ nor rescue agent, showing maximum cell viability. PQ-only wells (n = 32) show low viability in wells receiving only PQ. PQ + drug (N = 640) received PQ plus a test agent. Each symbol designates a different well. RLU, relative light units. (B) Histograms for tested agents (n = 6351), showing number of tested compounds providing specific degrees of cellular protection for PQ, Cd, and MMS. Scores have been normalized by adjustment for plate-to-plate variation in median cell viability and are expressed on a log10 scale. See Methods for details. Red arrows designate the 90th percentile for each distribution. (C) Proportional Venn diagram showing overlap among compounds in the top 10% for induction of PQ, Cd, and MMS resistance in primary screening. Figure generated using http://biovenn.nl/venndiagram.tk/create.php.

  • Fig. 2 DR curves for selected small molecules.

    (A) Representative DR curves, for concentrations from 32 to 0.25 μM (X axis). Green arrow designates mean score (from four daily batches) for wells with neither PQ nor protective agent added, and red arrow designates mean score for wells that received PQ alone. Gray dashed line shows a viability score of 100, used as an arbitrary criterion for categorizing an agent as protective. (B) Scatterplot for ED50 versus Max viability score for PQ protection; each symbol represents a different rescue agent. (C) Scatterplot showing Max score for PQ protection versus Max protection for Cd stress. Blue triangles indicate rescue agents for which Max > 100 in the MMS protection dose testing. Green dashed lines indicate arbitrary thresholds at Max = 100 for PQ and Cd protection scores.

  • Fig. 3 Tetracycline and dihydrorotenone increase lifespan in C. elegans.

    Representative Kaplan-Meier survival curves of C. elegans treated with tetracycline (A) or dihydrorotenone (B). Experimental replicates are shown as multiple lines.

  • Fig. 4 Transcriptomic analysis of eight rescue compounds.

    (A) PCA for all RNA-seq samples. (B) Dendrogram with unsupervised hierarchal clustering of the top 50 genes with greatest variance from the mean. Expression across each gene (row) has been scaled such that the mean expression is zero (pale yellow). Red indicates high expression in comparison to the mean. Blue indicates low expression in comparison to the mean. Lighter shades represent intermediate levels of expression. PC1, principal component 1.

  • Fig. 5 Nrf2 antioxidant signature in response to AEG 3482 or cardamonin treatment.

    (A) Pathways related to oxidative stress response from IPA for DE genes induced by AEG 3482 and cardominin. P values represented in negative log form; all pathways are significant at P < 0.05. eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric oxide synthase. (B) GSEA for cardamonin for pathways indicated. NES, normalized enrichment score. (C) Transcripts in key pathways induced by Nrf2 are significantly elevated by AEG 3482 and cardamonin treatment. All FDRs for comparison to DMSO are q < 5 × 10−6. TPM, transcripts per million. (D) Immunoblot analysis of Nrf2 targets after treatment with indicated compounds for 48 hours. PCNA and SDHA serve as loading controls. (E) Immunoblot in (D) was performed three times using independent biological samples. Band intensities were quantified by ImageJ and then normalized to control loading band (SDHA or PCNA). Mean and SEM are designated. Each treatment was compared to DMSO via two-tailed unpaired t test. *P < 0.05, **P < 0.01, and ****P < 0.0001. ns, not significant.

  • Fig. 6 Cardamonin and AEG 3482 promote SKN-1–dependent PQ resistance in C. elegans.

    (A) Increased PQ resistance conferred to C. elegans by cardamonin and AEG 3482 and three other compounds analyzed by RNA-seq. Log-rank P values versus PQ alone are as follows: 5 × 10−13 AEG 3482, 8 × 10−10 cardamonin, 6 × 10−9 dihydrorotenone, 7 × 10−5 berberine, 7 × 10−4 clofilium tosylate, 0.4 podophyllotoxin, 0.4 diphenyleneiodonium, and 0.01 antimycin A. (B) PQ, cardamonin, and AEG 3482, but not podophyllotoxin, induce the expression of a gst-4p::gfp reporter. Each point designates fluorescence in an individual worm treated as indicated. DMSO was compared to other treatments by one-way analysis of variance (ANOVA). See fig. S6 for two additional experimental replicates. ***P < 0.001 and **P < 0.01. (C) PQ resistance induced by cardamonin and AEG 3482 requires intact SKN-1 function. Log-rank P values are as follows: WT M9 v. WT + PQ 2 × 10−16; skn-1(−) M9 v. skn-1(−) + PQ 2 × 10−16; WT + PQ v. WT AEG 3842 + PQ 1 × 10−13; WT + PQ v. WT cardamonin + PQ 2 × 10−10; skn-1(−) + PQ v. skn-1(−) AEG 3482 + PQ 0.09; skn-1(−) + PQ v. skn-1(−) cardamonin + PQ 0.4.

Supplementary Materials

  • Supplementary Materials

    High-throughput small molecule screening reveals Nrf2-dependent and -independent pathways of cellular stress resistance

    David B. Lombard, William J. Kohler, Angela H. Guo, Christi Gendron, Melissa Han, Weiqiao Ding, Yang Lyu, Tsui-Ting Ching, Feng-Yung Wang, Tuhin S. Chakraborty, Zaneta Nikolovska-Coleska, Yuzhu Duan, Thomas Girke, Ao-Lin Hsu, Scott D. Pletcher, Richard A. Miller

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