Research Paper Advance Articles pp 23619—23646

Pathomechanism characterization and potential therapeutics identification for SCA3 targeting neuroinflammation

Figure 4. Effects of the tested compounds in ATXN3/Q75-GFP-expressing SH-SY5Y cells inflamed with IFN-γ-stimulated HMC3 conditioned medium. Experimental flowchart (A). ATXN3/Q75-GFP SH-SY5Y cells were plated in media with retinoic acid (RA, 10 μM) on day 1, and treated with the tested compound (10 μM) next day for 8 h, followed by doxycycline addition (Dox, 5 μg/ml) to induce ATXN3/Q75 expression. On day 6, DMEM-F12 medium without retinoic acid addition (− RA) was mixed with HMC3 conditioned medium with or without IFN-γ stimulation (CM/+IFN-γ or CM/–IFN-γ, 1:1 ratio) and added to the cells for 2 days. Cell viability, caspase 1 activity, LDH release (B), ROS (D), polyQ aggregation, neurite length, process and branch (F) were assessed on day 8 (n = 3). For normalization, the relative cell viability, caspase 1 activity, LDH release and ROS levels of cells treated with CM/–IFN-γ were set as 100%. (C) Images of ROS assay using CellROX dye (red).