Oxidative Medicine and Cellular Longevity / 2020 / Article / Fig 5

Research Article

New Synthetic 3-Benzoyl-5-Hydroxy-2H-Chromen-2-One (LM-031) Inhibits Polyglutamine Aggregation and Promotes Neurite Outgrowth through Enhancement of CREB, NRF2, and Reduction of AMPKα in SCA17 Cell Models

Figure 5

NRF2 and CREB as therapeutic targets in LM-031 and licochalcone A-treated TBP/Q79-GFP-expressing SH-SY5Y cells. (a) Experimental flow chart. On day 1, TBP/Q79-GFP SH-SY5Y cells were plated with retinoic acid (10 μM). On day 2, the cells were infected with lentivirus-expressing NRF2-specific, CREB-specific, or scrambled shRNA (MOI: 3). At 24 h postinfection, LM-031 or licochalcone A (100 nM) was added to the cells for 8 h, followed by induction of TBP/Q79-GFP expression (Dox, 5 μg/mL) for 6 days. On day 9, the cells were collected for NRF2 and pCREB protein analysis (by Western blot, GAPDH as a loading control) or stained with Hoechst 33342 for aggregation and neurite outgrowth analyses (by HCA). (b) Western blot analysis of NRF2 and pCREB protein levels in LM-031 or licochalcone A-treated cells infected with NRF2, CREB, or a negative control scramble (Scr.) shRNA. To normalize, the relative NRF2 or pCREB level of scramble shRNA-infected cells without inducing TBP/Q79-GFP expression was set as 100%. values: comparisons between induced versus uninduced cells (#, ###), compound-treated versus untreated cells (, ), or scramble versus NRF2 or CREB shRNA-infected cells (& or $: , &&& or $$$: ) (). (c) Aggregation and (d) neurite outgrowth assays of LM-031 or licochalcone A-treated TBP/Q79-GFP SH-SY5Y cells infected with NRF2, CREB, or a scramble shRNA. values: comparisons between compound-treated versus untreated cells (, ) or scramble shRNA versus NRF2 or CREB shRNA-infected cells (&&& or $$$: ) (). (e−f) Representative microscopy images of TBP/Q79-GFP-expressing SH-SY5Y cells infected with scrambled (Scr.), NRF2, or CREB shRNA, or LM-031 and licochalcone A (100 nM)-treated cells infected with scrambled, NRF2, or CREB shRNA. Aggregates were marked in red (e), and neurites and cell bodies were outlined (f) for outgrowth quantification.

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