Extended Data Fig. 4: High-throughput chemical screen quality control and validation of HDAC3.

a, Example images of DMSO vehicle-treated reactive and physiological control wells from the phenotypic screen. Scale bar is 100 µm. b, Z′ standard and robust scores for primary screen plates. c, Percent GBP2 positive astrocytes in DMSO vehicle treated reactive and physiological control wells on each primary screen plate. Data as the mean ± s.e.m., n = 16 wells per group on each primary screen plate. d, Dose curve of primary screen hits. Data are percent of GBP2 positive cells normalized to DMSO vehicle treated reactive astrocyte control wells. n = 2 biological replicates (independent astrocyte isolations). Black data points represent toxic doses where total cell number decreased by >50% compared to vehicle treated reactive astrocyte control wells. e, Dose curve analysis of hits from primary screen with Psmb8 positivity by in situ hybridization as a secondary endpoint. Data are percent of Psmb8 mRNA positive normalized to DMSO vehicle treated reactive astrocyte control wells with an n = 1 biological replicate (independent astrocyte isolation). Black data points represent toxic doses where total cell number decreased by >50% compared to vehicle treated reactive astrocyte control wells. f, Ranked inhibition against each HDAC isozyme for validated HDAC inhibitors in the primary screen. Highlighted is HDAC as the only shared target between all HDAC inhibitor hits. Ranked efficiency was pulled from target data provided by Selleck Chemical. g, Dose curve and IC50 value for the HDAC3 specific inhibitor RGFP966 to block astrocyte reactivity with an n = 5 biological replicates. h, Dose curve and IC50 value for the HDAC3 specific inhibitor T247 to block astrocyte reactivity with an n = 2 biological replicates. i–k, Representative images and quantification of wild-type (WT) and HDAC3 knockout (KO) astrocyte cultures exposed to TIC cytokines. Scale bar is 100 µm. Data are mean ± s.e.m., n = 3 independent experiments, p-value by a paired t-test. l, GBP2 and PSMB8 qPCR results for human iPSC derived physiological or reactive astrocyte cultures treated with vehicle or 5 μM RGFP966. Data are mean ± s.e.m., n = 4 technical replicates.

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