Extended Data Fig. 7: In vivo pharmacology of RGFP966 and in vivo genetic validation of HDAC3 as a mediator of astrocyte reactivity.
From: A phenotypic screening platform for identifying chemical modulators of astrocyte reactivity
a, Brain concentration of RGFP966 (RGFP) 4 hrs after treatment with vehicle (Veh) or 10 mg/kg RGFP. Data presented for n = 2 biological replicates (mice). Concentration of RGFP in brain from vehicle treated mice was below quantifiable levels (BQL). b–d, Representative images and quantification of immunohistochemistry for AcH4 in the cortex of mice treated with vehicle or 10 mg/kg RGFP and then exposed to systemic LPS injections to induce neuroinflammation. Scale bar is 100 µm. Data are mean ± s.e.m., n = 4 biological replicates (mice), p-value by unpaired two-tailed t-test e,f, Representative in situ hybridization images and quantification of untreated and LPS-exposed mice probed for the pan-reactive astrocyte marker Gfap (blue) and the reactive astrocyte marker Gbp2 (red) in the corpus callosum. Scale bar is 50 µm. Data are mean ± s.e.m., n = 4 biological replicates (mice), p-value by unpaired two-tailed t-test. g–m, Representative images and quantification of immunohistochemistry for GFAP (red) and IBA-1 (blue) in the cortex of mice treated with vehicle or 10 mg/kg RGFP and exposed to systemic LPS or saline vehicle. Scale bar is 100 µm. Data are mean ± s.e.m., n = 3 or 4 biological replicates (mice), p-value calculated by one-way ANOVA and Tukey multiple comparison correction. n, Diagram of astrocyte specific HDAC3 knockout mouse breeding. o–q, Representative in situ hybridization images and quantification of untreated wild-type (WT) and HDAC3 knockout (KO) mice and then probed for the pan-astrocyte marker Slc1a3 (green) and the reactive astrocyte marker C3 (red). Scale bar is 50 µm. Data are mean ± s.e.m., n = 3 biological replicates (mice), p-value by unpaired two-tailed t-test. r–u, Representative in situ hybridization images and quantification of wild-type and HDAC3 knockout (KO) mice exposed to systemic LPS and then probed for the pan-astrocyte marker Slc1a3 (green) and the reactive astrocyte marker C3 (red). Scale bar is 50 µm. Data are mean ± s.e.m., n = 4 biological replicates (mice), p-value by unpaired two-tailed t-test.
