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 2

Aggregation and ROS analyses on TBP/Q₇₉-GFP-expressing 293 cells. (a) Experimental flow chart. TBP/Q₇₉-GFP 293 cells were plated on dishes, grown for 24 h, and treated with SAHA (100 nM) or test compounds (0.1 nM−100 μM) for 8 h. Then, doxycycline (10 μg/mL) and oxaliplatin (5 μM) were added to the medium for 6 days, followed by aggregation (by HCA) and ROS (by flow cytometry) measurements. (b) Fluorescent microscopy image of cells with induced TBP/Q₇₉-GFP expression (green) for six days. Nuclei were counterstained with Hoechst 33342 (blue). White arrows indicate aggregates. (c) Representative microscopy images of TBP/Q₇₉-GFP cells untreated, or treated with licochalcone A or LM-031 (100 nM) for 6 days, with nuclei counterstained (blue, top row) or aggregates marked (red, bottom row). The magnified boxed area of the untreated TBP/Q₇₉-GFP-expressing 293 cells is displayed as (b). (d) Aggregation analysis () of TBP/Q₇₉-GFP-expressing cells untreated or treated with SAHA (100 nM) or test compounds (0.1 nM−100 μM). To normalize, the relative aggregation level in untreated cells was set as 100%. The red line represents 82% aggregation for 100 nM SAHA treatment. values: comparisons between test-compounds treated and SAHA treated (, , ). Aggregation was analyzed in wells containing at least 80% viable cells. (e) The induced GFP and ROS levels were measured by flow cytometry (). values: comparisons between induced and uninduced cells (^###), or between compound (100 nM) treated and untreated cells ().

(a)

(b)

(c)

(d)

(e)