Research Paper Volume 15, Issue 16 pp 8061—8089

Figure 5. α-Synuclein aggregation reduction and neurite outgrowth promotion of LM-021 and NC009-1 on A53T-8 SNCA-GFP BE(2)-M17 cells. The cells were added with retinoic acid on day 1, and treated with compound, doxycycline and α-synuclein fibril on day 2. On day 7, α-synuclein aggregation and neurite outgrowth were assessed. (A) α-Synuclein aggregation analyzed by filter trap assay detected by using a GFP antibody (n = 3). To normalize, the α-synuclein aggregates with fibril addition was set as 100%. (B) Fluorescent microscopy images of SNCA-GFP-expressing cells (green) with or without preformed fibril addition or compound treatment and quantification of percentage of aggregated cells (n = 3). Nuclei were detected with Hoechst 33342 (blue) and overlapped SNCA-GFP and ProteoStat®-stained (red) aggregates marked (yellow). (C) Fluorescent microscopy images of SNCA-GFP cells stained with TUBB3 for quantifying neurite outgrowth with or without preformed fibril addition or compound treatment, with nuclei detected (DAPI, blue). Also shown were images of the neurites and cell bodies being outlined with different colors to indicate different neurons for quantifying neurite outgrowth. In uninduced cells, processes and branches are indicated with red and white arrows, respectively. Quantification of neurite length, brunch and process was shown below (n = 3). P values: with vs. without doxycycline addition (^##: P < 0.01, ^###: P < 0.001), with vs. without fibrils addition (^&&: P < 0.01, ^&&&: P < 0.001), or with vs. without compound treatment (**: P < 0.01, ***: P < 0.001).