Research Paper Volume 11, Issue 3 pp 986—1007

Figure 4. PGC-1α, NRF2, and NFYA as therapeutic targets in SG-Tang-treated TBP/Q₇₉-GFP-expressing SH-SY5Y cells. (A) Experimental flowchart. TBP/Q₇₉-GFP SH-SY5Y cells were plated on dishes with retinoic acid (RA, 10 µM) added at day 1. Next day, cells were transfected with siRNA (100 nM; PGC-1α, NRF2, NFYA, or scrambled control). At 24 h post-transfection, SG-Tang (100 µM) was added to the cells for 8 h followed by inducing TBP/Q₇₉-GFP expression (+ Dox, 5 µg/ml) for 6 days. Then, the cells were collected for PGC-1α, NRF2, and NFYA protein analysis (by Western blot, GAPDH as a loading control), or stained with Hoechst 33342 for aggregation, neurite outgrowth, process, and branch analyses (by HCA). (B) Western blot analysis of PGC-1α, NRF2, and NFYA protein levels in SG-Tang-treated cells transfected with PGC-1α, NRF2, NFYA, or scrambled control siRNA. To normalize, the relative PGC-1α, NRF2, or NFYA level of uninduced cells was set as 100%. P values: induced versus uninduced cells (^#: P < 0.05 and ^##: P < 0.01); SG-Tang-treated versus untreated cells (*: P < 0.05 and **: P < 0.01); or PGC-1α, NRF2, or NFYA siRNA versus scrambled control siRNA-transfected cells (^&: P < 0.05) (n = 3). (C) Representative microscopic images of TBP/Q₇₉-GFP-expressing SH-SY5Y cells transfected with scrambled control siRNA and SG-Tang-treated cells transfected with scrambled control, PGC-1α, NRF2, or NFYA siRNA. Nuclei were counterstained with Hoechst 33342 (blue, left) and aggregates were marked in red (middle). Shown left are images of the neurites and cell bodies outlined by red color for neurite outgrowth, process, and branch quantification. (D) Aggregation and neuronal outgrowth, process, and branch assays of SG-Tang-treated TBP/Q₇₉-GFP-expressing SH-SY5Y cells transfected with scrambled control, PGC-1α, NRF2, or NFYA siRNA. To normalize, the relative aggregation, outgrowth, process, or branch of scrambled control siRNA-transfected cells without SG-Tang treatment was set as 100%. P values: SG-Tang-treated versus untreated cells (*: P < 0.05); PGC-1α, NRF2, or NFYA siRNA versus scrambled control siRNA-transfected cells (^&: P < 0.05 and ^&&: P < 0.01) (n = 3).